U.S. patent application number 10/481090 was filed with the patent office on 2005-07-21 for extracellular junctional adhesion molecules.
Invention is credited to Heuer, Josef Georg, Smith, Rosamund Carol, Su, Eric Wen.
Application Number | 20050159587 10/481090 |
Document ID | / |
Family ID | 26974764 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050159587 |
Kind Code |
A1 |
Heuer, Josef Georg ; et
al. |
July 21, 2005 |
Extracellular junctional adhesion molecules
Abstract
This invention provides human extracellular junctional adhesion
molecules (huJAM) and polynucleotides which identify and encode
huJAM. The invention further provides methods using the molecules
of the invention for treating, cancer and inflammatory, immune
system, and cardiovascular disorders.
Inventors: |
Heuer, Josef Georg;
(Indianapolis, IN) ; Smith, Rosamund Carol;
(Greenfield, IN) ; Su, Eric Wen; (Carmel,
IN) |
Correspondence
Address: |
ELI LILLY AND COMPANY
PATENT DIVISION
P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
26974764 |
Appl. No.: |
10/481090 |
Filed: |
December 16, 2003 |
PCT Filed: |
July 5, 2002 |
PCT NO: |
PCT/US02/19800 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60305752 |
Jul 16, 2001 |
|
|
|
60354345 |
Feb 5, 2002 |
|
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Current U.S.
Class: |
530/350 ;
424/143.1; 435/320.1; 435/325; 435/69.1; 530/388.22; 536/23.5 |
Current CPC
Class: |
C12N 2799/026 20130101;
C07K 2319/00 20130101; C07K 2319/30 20130101; C07K 14/70503
20130101 |
Class at
Publication: |
530/350 ;
424/143.1; 435/069.1; 435/320.1; 435/325; 530/388.22;
536/023.5 |
International
Class: |
C07K 014/705; C07K
016/28; C12P 021/02; A61K 039/395 |
Claims
1. A purified extracellular huJAM polypeptide comprising at least
95% sequence identity to an amino acid sequence selected from the
group consisting of SEQ ID NOS: 8, 11, 12, and 14.
2. The extracellular huJAM polypeptide of claim 1, wherein said
polypeptide lacks a transmembrane domain.
3. The extracellular huJAM polypeptide of claim 2, wherein said
polypeptide is capable of binding a huJAM ligand.
4. The purified extracellular huJAM polypeptide of claim 2 encoded
by a polynucleotide, wherein said polynucleotide lacks sequence
encoding a transmembrane domain.
5. A purified extracellular huJAM polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NOS: 8,
11, 12, and 14.
6. The extracellular huJAM polypeptide of claim 5, wherein said
polypeptide lacks a transmembrane domain.
7. The extracellular huJAM polypeptide of claim 6, wherein said
polypeptide is capable of binding a huJAM ligand.
8. The extracellular huJAM polypeptide of claim 6 encoded by a
polynucleotide, wherein said polynucleotide lacks sequence encoding
a transmembrane domain.
9. An isolated polynucleotide encoding an extracellular huJAM
polypeptide and having at least 95% sequence identity to a nucleic
acid sequence selected from the group consisting of SEQ ID NOS: 15,
16, 17, 18, 19, 20, 21, and 22.
10. The polynucleotide of claim 9 wherein said polynucleotide lacks
sequence encoding a transmembrane domain.
11. An isolated polynucleotide encoding an extracellular huJAM
protein wherein said polynucleotide is selected from the group
consisting of SEQ ID NOS: 15, 16, 17, 18, 19, 20, 21, and 22.
12. A vector comprising a polynucleotide encoding an extracellular
huJam protein as the sole source of huJAM nucleotide sequence.
13. The vector of claim 12, which is an expression vector.
14. A host cell transfected with the vector of claim 12.
15. A host cell transfected with the expression vector of claim
13.
16. The host cell of claim 15, wherein said cell is a CHO cell.
17. The host cell of claim 15, wherein said cell is an E. coli
cell.
18. The host cell of claim 15, wherein said cell is an Sf9
cell.
19. The host cell of claim 15, wherein said cell is a yeast
cell.
20. A method for producing a polypeptide, the method comprising the
steps of: a) culturing the host cell of claim 15 under conditions
suitable for the expression of the polypeptide from the said
polynucleotide and b) recovering the polypeptide from the cell
culture medium.
21. An isolated polypeptide produced by the method of claim 20.
22. A chimeric molecule comprising the extracellular huJAM of claim
1 fused to a heterologous amino acid sequence wherein the
extracellular huJAM of claim 1 is the sole source of huJAM.
23. The chimeric molecule of claim 22, wherein said heterologous
amino acid sequence is an epitope tag sequence.
24. The chimeric molecule of claim 22, wherein said heterologous
amino acid sequence is an Fc region of an immunoglobulin.
25. A chimeric molecule comprising the extracellular huJAM of claim
5 fused to a heterologous amino acid sequence wherein the
extracellular huJAM of claim 5 is the sole source of huJAM.
26. The chimeric molecule of claim 22, wherein said heterologous
amino acid sequence is an epitope tag sequence.
27. The chimeric molecule of claim 22, wherein said heterologous
amino acid sequence is an Fc region of an immunoglobulin.
28. A pharmaceutical composition comprising the extracellular huJAM
of claim 1 in conjunction with a suitable pharmaceutical
carrier.
29. A pharmaceutical composition comprising the extracellular huJAM
of claim 5 in conjunction with a suitable pharmaceutical
carrier.
30. A method of treating an immune system disorder comprising
administering a therapeutically effective amount of the
extracellular huJAM of claim 1 to a mammal having said
disorder.
31. The method of claim 30, wherein the immune system disorder is
an immune deficiency, an autoimmune disease or an inflammatory
disorder.
32. A method of treating an immune system disorder comprising
administering a therapeutically effective amount of the
extracellular huJAM of claim 5 to a mammal having said
disorder.
33. The method of claim 32, wherein the immune system disorder is
an immune deficiency, an autoimmune disease or an inflammatory
disorder.
37. A method of treating cancer comprising administering a
therapeutically effective amount of an extracellular huJAM of claim
1 to a mammal having said cancer.
38. A method of treating a cardiovascular disorder comprising
administering a therapeutically effective amount of an
extracellular huJAM of claim 1 to a mammal having said
disorder.
39. A method of treating wound healing comprising administering a
therapeutically effective amount of an extracellular huJAM of claim
1 to a mammal in need of such treatment.
40. An article of manufacture comprising a container, label and
therapeutically effective amount of an extracellular huJAM of claim
1 in combination with a pharmaceutically effective carrier.
Description
FIELD OF THE INVENTION
[0001] This invention relates to human extracellular junctional
adhesion molecules (JAM) and polynucleotides which identify and
encode human extracellular JAM. The invention further provides
compositions and methods using the proteins and polynucleotides of
the invention for treating cancer, cardiovascular disorders, and
immune system disorders such as autoimmune diseases and
inflammatory disorders.
BACKGROUND OF THE INVENTION
[0002] Junctional adhesion molecules (JAM) are members of the
immunoglobulin superfamily (IgSf). JAM have two extracellular IgSf
domains, a transmembrane segment, and a short cytoplasmic segment.
Currently, three distinct JAM proteins, JAM1, JAM2, and JAM3, have
been identified in both murine and human sources. JAM are localized
to the intercellular boundaries of endothelial and epithelial cells
although the tissue distribution pattern for each is distinct
(Aurrand-Lions, M. et al. Curr. Top. Microbiol. Immunol. 251:
91-98, 2000).
[0003] Endothelial cells lining blood vessels form a blood-tissue
barrier and such cells are attached to each other by at least two
types of complex, junctional structures, adherens junctions (AJ)
and tight junctions (TJ), to form a continuous layer of cells.
JAM1, JAM2, and JAM3 are concentrated at the sites of cell-cell
junctions for both endothelial and epithelial cells and facilitate
cell-cell contact through homotypic and/or heterotypic
interactions. JAM are also functionally implicated in cell
trafficking and cell-fate determination (Malergue, F. et al. Mol.
Immunol. 35: 1111-1119, 1998).
[0004] Leukocytes, it is commonly believed, leave the blood by
first adhering to endothelial cells and then migrating through the
interendothelial junctions. In doing so they cause disruption of
the junctional structures. The process by which leukocytes traverse
these junctions is not completely understood, particularly on a
molecular level. However, it has been proposed that JAM play a
structural role in the control of leukocyte migration across
epithelium or endothelium to sites of inflammation. JAM1, localized
to TJ, is involved in myeloid cell and neutrophil transmigration
(Padura, I., et al. J. Cell Biol. 142: 117-127, 1998) while JAM2
may have a role in controlling leukocyte recirculation at secondary
lymphoid organs such as lymph nodes and tonsils (Aurrand-Lions, M.
et al. Curr. Top. Microbiol. Immunol. 251: 91-98, 2000). Much
remains to be learned about the role of JAM in leukocyte
infiltration and in the inflammation process.
[0005] Human JAM (huJAM) is reported to be expressed in circulating
immune cells at high levels, both at the mRNA and at the protein
level (Williams, L., et al., Mol. Immunology 36: 1175-1188, 1999).
It is contemplated that a JAM polypeptide on the endothelial cell
interacts with a JAM polypeptide on the immune cell thereby
inducing transmembrane signaling and the passage of immune cells
through the interendothelial junction.
[0006] Numerous publications and databases have reported the
full-length, membrane-bound sequence of murine (mu) and human (hu)
JAM1, JAM2, and JAM3 polypeptides as well as the polynucleotide
sequences encoding the JAM polypeptides (e.g., International patent
publications: WO9842739, WO9840483, WO9927098, WO9914241,
WO0073452, WO0029583, WO0061623, WO0053758, WO0053749, WO0056754,
WO0053758, WO0107459). While WO0053758 (page 6) makes mention of a
transmembrane-deleted human JAM3, it does not identify the location
or sequence of the transmembrane sequence nor does it identify a
function of such a molecule that is distinct from that of the
molecule containing the transmembrane domain.
[0007] It is well known that the regulated and coordinated
expression of adhesion molecules is required for normal vascular
function. During inflammation, the cell-cell interactions of the
epithelial cell layer are disrupted, resulting in a leaky
epithelial barrier, which in turn can lead to various inflammatory
and infective disorders. Changes in the adhesion properties of
vascular endothelial cells are also observed during tumor growth,
wound healing, and angiogenesis. There is great clinical potential
and need for extracellular junction adhesion molecules which can
function as agonists and/or antagonists and thereby prevent
leukocyte transmigration across adherens junctions or tight
junctions and be useful for the diagnosis, prevention and treatment
of cancer, cardiovascular disorders, and immune system
disorders.
SUMMARY OF THE INVENTION
[0008] The present invention addresses the need for human JAM
(huJAM) agonists and/or antagonists by providing extracellular
huJAM polypeptides and related compositions and methods.
[0009] The present invention embodies extracellular huJAM
polypeptides (LP121A (SEQ ID NO:11), LP121B (SEQ ID NO:12), LP121C
(SEQ ID NO:8), LP10034 (SEQ ID NO:14)) and their use in treating
cancer, cardiovascular disorders, and immune system disorders such
as autoimmune diseases and inflammatory disorders.
[0010] Amino acid residues 1 to about 30 of the full-length huJAM
proteins (SEQ ID NOS: 1, 2, and 3) are a signal peptide that is
removed by a signal peptidase enzyme during maturation of the huJAM
protein. While extracellular huJAM polypeptides LP121A, LP121B,
LP121C and LP10034 can be encoded by a nucleic acid that encodes
the signal peptide (SEQ ID Nos: 15, 16, 17, and 18), this signal
peptide is cleaved off and is not present in the mature,
extracellular form of the protein. Alternatively, the extracellular
huJAM polypeptides LP121A, LP121B, LP121C and LP10034 can be
encoded by a nucleic acid that lacks sequence encoding the signal
peptide (SEQ ID Nos: 19, 20, 21, and 22). Also lacking in LP121A,
LP121B, LP121C and LP10034 polypeptides, regardless of whether or
not the signal peptide is present, are the transmembrane domain and
cytoplasmic domain that are present in full-length, membrane-bound
huJAM (see FIG. 1 for the domain boundaries and Table 2 for
nomenclature summary).
[0011] The invention embodies multiple forms of isolated and
purified extracellular huJAM polypeptides e.g., extracellular
huJAM2 also referred to herein as LP121A (SEQ ID NO:11) and LP121B
(SEQ ID NO:12); extracellular huJAM1 also referred to herein as
LP121C (SEQ ID NO:8); and extracellular huJAM3 also referred to
herein as LP10034 (SEQ ID:14). The invention further embodies
extracellular huJAM polypeptide variants with an amino acid
sequence variation of SEQ ID NOS: 8, 11, 12, or 14 as further
described herein. It is contemplated that the extracellular huJAM3
of the invention embodies allelic variants e.g., the variant in
which the amino acid at position 195 of the full length protein,
(SEQ ID NO:3), or the equivalent position in the mature protein, is
either a phenylalanine or a serine. The invention further
contemplates LP polypeptides that have an amino acid sequence at
least about 95%, even more preferably at least 96%, 97%, or 98% and
most preferably at least 99% identical (i.e., amino acid sequence
identity) to that shown in SEQ ID NOS: 8, 11, 12, or 14.
[0012] The invention also embodies an expression vector encoding an
extracellular huJAM polypeptide, or an extracellular huJAM
polypeptide variant, operably linked to a promoter sequence and a
host cell transfected with such an expression vector that produces
an extracellular huJAM polypeptide. Exemplary host cells include,
but are not limited to, CHO cells, E. coli cells, Sf9 cells and
yeast cells. The type of promoter sequence used in the expression
vector will vary depending upon the host cell type.
[0013] A further aspect of the invention embodies an isolated and
purified fusion polypeptide consisting essentially of a first
portion and a second portion joined by a peptide bond. The first
portion of the fusion polypeptide comprises (a) an extracellular
huJAM with an amino acid sequence shown in SEQ ID NOS: 8, 11, 12,
or 14 as the sole source of huJAM in the fusion polypeptide, (b) an
extracellular huJAM variant with an amino acid sequence variation
of the sequence shown in SEQ ID NOS: 8, 11, 12, or 14 as the sole
source of huJAM in the fusion polypeptide or (c) a protein
polypeptide that has an amino acid sequence at least about 95%,
even more preferably at least 96%, 97%, or 98% and most preferably
at least 99% identical (i.e., amino acid sequence identity) to that
described in (a) or (b). The second portion of the fusion
polypeptide consists of another polypeptide such as an affinity
tag. Within one embodiment the affinity tag is an immunoglobulin Fc
polypeptide. Within another embodiment the affinity tag is FLAG or
His6.
[0014] The invention also provides an expression vector encoding a
fusion polypeptide and a host cell transfected with such an
expression vector to produce a fusion polypeptide wherein the
fusion polypeptide consists essentially of a first portion and a
second portion joined by a peptide bond. The first portion of the
fusion polypeptide comprises (a) an extracellular huJAM with an
amino acid sequence shown in SEQ ID NOS: 8, 11, 12, or 14 as the
sole source of huJAM in the fusion polypeptide, (b) an
extracellular huJAM variant with an amino acid sequence variation
of the sequence shown in SEQ ID NOS: 8, 11, 12, or 14 as the sole
source of huJAM in the fusion polypeptide or (c) a protein
polypeptide with an amino acid sequence that is at least about 95%,
even more preferably at least about 96%, 97%, or 98% and most
preferably at least 99% identical (i.e., amino acid sequence
identity) to that described in (a) or (b). The second portion of
the fusion polypeptide consists of another polypeptide such as an
affinity tag. Within one embodiment the affinity tag is an
immunoglobulin Fc polypeptide. Within another embodiment the
affinity tag is FLAG or His6. Exemplary host cells include, but are
not limited to, CHO cells, E. coli cells, Sf9 cells and yeast
cells.
[0015] One embodiment of the invention provides isolated nucleic
acid molecules encoding a polypeptide of the present invention
including mRNAs, DNAs, cDNAs, and genomic DNAs.
[0016] The present invention also provides isolated and purified
polynucleotides encoding an extracellular huJAM. More specifically,
such polynucleotides have the DNA sequence shown in (a) SEQ ID NOS:
15, 16, 17, and 18, (b) complements of SEQ ID NOS: 15, 16, 17, and
18, (c) isolated polynucleotides, preferably DNA, that hybridize to
(a) or (b) and not to a huJAM transmembrane domain (as identified
in FIGS. 1-6 herein), under stringent hybridization and wash
conditions, and (d) nucleic acid molecules, preferably DNA,
comprising a nucleotide sequence having at least 90%, 91%, 92%,
93%, or 94% or more preferably at least 95%, 96%, 97%, or 98% or
most preferably at least 99% nucleic acid sequence identity to a
nucleic acid molecule of (a), (b), or (c).
[0017] While SEQ ID NOS: 15, 16, 17, and 18 have nucleic acid
sequence encoding the signal peptide, it is contemplated that
polynucleotides encoding an extracellular huJAM can lack the
nucleic acid sequence encoding the signal peptide and fall within
the bounds of the invention. Such polynucleotides have the DNA
sequence shown in (e) SEQ ID NOS: 19, 20, 21, and 22, (f)
complements of SEQ ID NOS: 19, 20, 21, and 22, (g) polynucleotides,
preferably DNA, that hybridize to (e) or (f) and not to a huJAM
transmembrane domain under stringent hybridization and wash
conditions, and (h) nucleic acid molecules, preferably DNA,
comprising a nucleotide sequence having at least about 95%, 96%,
97%, or 98% or most preferably at least 99% nucleic acid sequence
identity to a nucleic acid molecule of (e), (f), or (g).
[0018] Additional compositions of the invention are those
comprising: (a) a purified, therapeutically effective,
extracellular huJAM polypeptide with an amino acid sequence shown
in SEQ ID NOS: 8, 11, 12, or 14 or variants thereof as the sole
source of huJAM sequence; or a purified, therapeutically effective
extracellular huJAM polypeptide with an amino acid sequence that is
at least about 95%, even more preferably at least 96%, 97%, or 98%
and most preferably at least 99% identical to the amino acid
sequence shown in SEQ ID NOS: 8, 11, 12, or 14 as the sole source
of huJAM sequence; or a purified, therapeutically effective fusion
protein comprising SEQ ID NOS: 8, 11, 12, or 14 or variations
thereof as the sole source of huJAM sequence and (b) a sterile
binding compound or binding agent, or the binding compound or
binding agent and a carrier, wherein the carrier is: an aqueous
compound including water, saline, and/or buffer; and formulated for
oral, rectal, nasal, topical, or parenteral administration. As used
herein "as the sole source of huJAM sequence" means that there is
no transmembrane domain, cytoplasmic domain, and no signal peptide
sequence present that originates from huJAM. It is, however,
contemplated that a composition can comprise one, two, or more
different extracellular huJAM.
[0019] In other embodiments, the invention provides a method of
modulating the physiology or development of a cell in vivo or in
situ comprising introducing into such cell, or the environment of
such cell, a therapeutically effective amount of LP121A (SEQ ID NO:
11), LP121B (SEQ ID NO: 12), LP121C (SEQ ID NO: 8), or LP10034 (SEQ
ID NO: 14) or a variant thereof, or a fusion protein comprising
LP121A (SEQ ID NO:11), LP121B (SEQ ID NO: 12), LP121C (SEQ ID NO:
8), or LP10034 (SEQ ID NO: 14), or a variant thereof, as the sole
source of huJAM in the fusion protein.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 provides an alignment of full-length membrane-bound
huJAM1 (SEQ ID NO: 1), huJAM2 (SEQ ID NO: 2) and huJAM3 (SEQ ID NO:
3) polypeptide sequences with consensus amino acids, putative
signal sequences, transmembrane domains and cytoplasmic domains
identified.
[0021] FIG. 2A provides the polynucleotide sequence encoding huJAM1
(SEQ ID NO: 4) and FIG. 2B provides the amino acid sequence of
full-length huJAM1 (SEQ ID NO: 1). The signal peptide and the
nucleic acid sequence encoding it are in bold, the transmembrane
and cytoplasmic regions 3' to the extracellular domain and the
nucleic acid sequence encoding it are underlined.
[0022] FIG. 3A provides the polynucleotide sequence encoding huJAM2
(SEQ ID NO: 5) and FIG. 3B provides the amino acid sequence of
full-length huJAM2 (SEQ ID NO:2). The signal peptide and the
nucleic acid sequence encoding it are in bold, the transmembrane
and cytoplasmic regions 3' to the extracellular domain and the
nucleic acid sequence encoding it are underlined.
[0023] FIG. 4A provides the polynucleotide sequence encoding huJAM3
(SEQ ID NO: 6) and FIG. 4B provides the amino acid sequence of
full-length huJAM3 (SEQ ID NO: 3). The signal peptide and the
nucleic acid sequence encoding it are in bold, the transmembrane
and cytoplasmic regions 3' to the extracellular domain and the
nucleic acid sequence encoding it are underlined.
[0024] FIG. 5 provides the amino acid sequence of the signal
peptide and extracellular domain of huJAM1 comprising amino acids
1-235 of full-length huJAM 1 (SEQ ID NO: 7).
[0025] FIG. 6 provides the amino acid sequence of the extracellular
domain of huJAM1 comprising amino acids 28-235 of full-length
huJAM1 (SEQ ID NO: 8).
[0026] FIG. 7A provides the amino acid sequence of the signal
peptide and extracellular domain of huJAM2 comprising amino acids
1-224 of the full-length huJAM2 (SEQ ID NO: 9).
[0027] FIG. 7B provides the amino acid sequence of the signal
peptide and extracellular domain of huJAM2 comprising amino acids
1-236 of the full-length huJAM2 (SEQ ID NO: 10).
[0028] FIG. 8A provides the amino acid sequence of the
extracellular domain of huJAM2 comprising amino acids 29-224 of the
full-length huJAM2 (SEQ ID NO: 11).
[0029] FIG. 8B provides the amino acid sequence of the
extracellular domain of huJAM2 comprising amino acids 29-236 of the
full-length huJAM2 (SEQ ID NO: 12).
[0030] FIG. 9 provides the amino acid sequence of the signal
peptide and extracellular domain of huJAM3 comprising amino acids
1-240 of the full-length huJAM3 (SEQ ID NO: 13).
[0031] FIG. 10 provides the amino acid sequence of the
extracellular domain of huJAM3 comprising amino acids 31-240 of the
full-length huJAM3 (SEQ ID NO: 14).
[0032] FIG. 11 provides the nucleotide sequence encoding the signal
peptide and extracellular domain of huJAM1 (amino acids 1-235 of
SEQ ID NO: 1)(SEQ ID NO: 15).
[0033] FIG. 12A provides the nucleotide sequence encoding the
signal peptide and extracellular domain of huJAM2 (amino acids
1-224 of SEQ ID NO: 2) (SEQ ID NO: 16).
[0034] FIG. 12B provides the nucleotide sequence encoding the
signal peptide and extracellular domain of huJAM2 (amino acids
1-236 of SEQ ID NO: 2) (SEQ ID NO: 17).
[0035] FIG. 13 provides the nucleotide sequence encoding the signal
peptide and extracellular domain of huJAM3 (amino acids 1-240 of
SEQ ID NO: 3) (SEQ ID NO: 18).
[0036] FIG. 14 provides the nucleotide sequence encoding the
extracellular domain of huJAM1 (amino acids 28-235 of SEQ ID NO: 1)
(SEQ ID NO: 19).
[0037] FIG. 15A provides the nucleotide sequence encoding the
extracellular domain of huJAM2 (amino acids 29-224 of SEQ ID NO: 2)
(SEQ ID NO: 20).
[0038] FIG. 15B provides the nucleotide sequence encoding the
extracellular domain of huJAM2 (amino acids 29-236 of SEQ ID NO: 2)
(SEQ ID NO: 21).
[0039] FIG. 16 provides the nucleotide sequence encoding the
extracellular domain of huJAM3 (amino acids 31-240 of SEQ ID NO: 3)
(SEQ ID NO: 22).
[0040] FIG. 17 provides the percent survival of mice injected with
LP121A and challenged with a lethal dose of LPS.
[0041] FIG. 18 provides murine serum TNF-.alpha. levels two hours
after mice injected with LP121A are challenged with a lethal dose
of LPS.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention is not limited to the particular embodiments
described below, as variations of the particular embodiments may be
made and still fall within the scope of the appended claims. The
terminology used herein is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
[0043] In this specification and the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0044] Definitions
[0045] To facilitate understanding of the invention, the following
terms are defined.
[0046] The terms "LP polypeptide(s)" and "LP" as used herein refer
to various polypeptides. The complete designation of LP immediately
followed by a number (LP121A, LP121B, LP121C, LP10034) refers to a
particular polypeptide sequence as described herein. The LP
polypeptides described herein may be isolated from a variety of
sources including, but not limited to, tissue culture media of
mammalian cells expressing the LP polypeptide, lysed E. coli
expressing the LP polypeptide, yeast, or Sf9 cells expressing the
LP polypeptide, or prepared by recombinant or synthetic
methods.
[0047] The term "isolated" when used in relation to a nucleic acid
or protein, means the material is identified and separated from at
least one contaminant with which it is ordinarily associated in its
natural source. Such a nucleic acid could be part of a vector
and/or such nucleic acid or protein could be part of a composition,
and still be isolated in that such vector or composition is not
part of its natural environment.
[0048] As used herein, the term "purified" means the result of any
process that removes from a sample a contaminant from the component
of interest, such as a protein or nucleic acid. The percent of a
purified component is thereby increased in the sample.
[0049] As used herein, the term "in situ" is used in reference to
activities, methods, functions and the like that occur in cell
culture conditions while the term "in vivo" is used in reference to
activities, methods, functions and the like that occur in an
organism.
[0050] As used herein, the terms "complementary" or
"complementarity" are used in reference to nucleic acids (i.e., a
sequence of nucleotides) related by the well-known base-pairing
rules that A pairs with T and C pairs with G. For example, the
sequence 5'-A-G-T-3', is complementary to the sequence 3'-T-C-A-5'.
Complementarity between two single-stranded molecules can be
"partial," in which only some of the nucleic acid bases are matched
according to the base pairing rules. On the other hand, there may
be "complete" or "total" complementarity between the nucleic acid
strands when all of the bases are matched according to base pairing
rules. The degree of complementarity between nucleic acid strands
has significant effects on the efficiency and strength of
hybridization between nucleic acid strands as known well in the
art.
[0051] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acid strands.
Hybridization and the strength of hybridization (i.e., the strength
of the association between nucleic acid strands) is impacted by
many factors well known in the art including the degree of
complementarity between the nucleic acids, level of stringency
involved is affected by such conditions as the concentration of
salts, the Tm (melting temperature) of the formed hybrid, the
presence of other components (e.g., the presence or absence of
polyethylene glycol), the molarity of the hybridizing strands and
the G:C content of the nucleic acid strands.
[0052] As used herein, the term "stringency" is used in reference
to the conditions of temperature, ionic strength, and the presence
of other compounds, under which nucleic acid hybridizations are
conducted. Hybridization generally depends on the ability of
denatured DNA to reanneal when complementary strands are present in
an environment below their melting temperature. With "high
stringency" or "highly stringent" or "stringent" conditions,
nucleic acid base pairing will occur only between nucleic acid
fragments that have a high frequency of complementary base
sequences. The art knows well that numerous equivalent conditions
can be employed to comprise high stringency conditions. "Stringent
conditions" or "high stringency conditions", as defined herein, are
identified by those that (1) employ low ionic strength and high
temperature for washing. The higher the degree of desired homology
between the two nucleic acid strands being hybridized, the higher
the relative temperature that can be used. As a result, it follows
that higher relative temperatures would tend to make the reactions
more stringent, while lower temperatures less so. For additional
details and explanation of stringency of hybridization reactions,
see Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, 1995 and supplements. Exemplary "high
stringency" or "stringent" conditions include hybridization
conditions of an overnight incubation of the two denatured nucleic
acid strands at 42.degree. C. in a solution comprising 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared, salmon sperm
DNA, followed by wash conditions of 68.degree. C. in the presence
of about 0.2.times.SSC and about 0.1% sodium dodecyl sulfate (SDS),
for one hour. (Reagents available from Sigma Corp., St. Louis, Mo.)
SSC concentration may be varied from about 0.1.times. to
2.0.times.SSC, with SDS optionally being present at about 0.1%
(w/v). Typically, blocking reagents are used to block non-specific
hybridization. Such blocking reagents include, for instance,
denatured salmon sperm DNA at about 100-200 .mu.g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% (v/v)
may also be used under particular circumstances, such as for
RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily apparent to those of ordinary skill in the art.
Hybridization, particularly under stringent conditions, may be
suggestive of evolutionary similarity between the nucleic acids.
Such similarity is strongly indicative of a similar role for the
nucleic acid molecules and the polypeptides they encode.
[0053] The term "homology," as used herein, refers to a degree of
complementarity. There can be partial homology or complete homology
(i.e., identity). A partially complementary sequence that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid is referred to using the
functional term "substantially homologous."
[0054] In the present invention "extracellular huJAM" refers to a
form of the said human JAM polypeptide which is essentially free of
the signal peptide and the transmembrane and cytoplasmic domains of
the full-length huJAM polypeptide. The exact boundaries of where
the signal peptide ends and the extracellular domain begins and the
exact boundaries of where the extracellular domain ends and the
transmembrane domain begins may vary but most likely by no more
than about six amino acids at either end of the domain as
identified herein. Therefore, an extracellular huJAM signal
peptide/extracellular domain boundary as identified in the
Examples, Figures, or specification may be shifted in either
direction (upstream or downstream) by 6, 5, 4, 3, 2, 1, or 0 amino
acids. Additionally, an extracellular huJAM extracellular
domain/transmembrane domain boundary as identified in the Examples
or specification may be shifted in either direction by 6, 5, 4, 3,
2, 1 or 0 amino acids. All such polypeptides and the nucleic acid
molecules encoding them are contemplated by the present invention.
For example, extracellular HuJAM1 domain is contemplated to extend
from amino acids 28-235 of the full-length HuJAM1 polypeptide (SEQ
ID NO:1), but it is contemplated that extracellular HuJAM1 could
span from an amino-terminal amino acid chosen from between amino
acids 22 through 34 ((inclusive) of the full-length HuJAM1 through
a carboxy-terminal amino acid from between amino acids 229 through
241 (inclusive) of the full-length HuJAM1.
[0055] "Conservative amino acid substitutions" are those
substitutions that, when made, least interfere with the properties
of the original protein, i.e., the structure and especially the
function of the protein is conserved and not significantly changed
by such substitutions. Table 1 below shows preferred conservative
amino acid substitutions for an original amino acid in a protein
with the most preferred substitution in bold type.
1 TABLE 1 Original Residue Conservative Substitution Ala (A) Val,
Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D)
Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala, Pro His (H)
Arg, Asn, Gln, Lys Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu
(L) Ile, norleucine, Val Met, Ala, Phe Lys (K) Arg, Gln, Asn Met
(M) Leu, Ile, Phe Phe (F) Leu, Val, Ile, Ala, Try Pro (P) Ala Ser
(S) Thr Thr (T) Ser Trp (W) Tyr, Phe Tyr (Y) Phe, Trp, Thr, Ser Val
(V) Leu, Ile, norleucine, Ala Phe, Met
[0056] Conservative amino acid substitutions generally maintain (a)
the structure of the polypeptide backbone in the area of the
substitution, for example, as a beta sheet or alpha helical
conformation, (b) the charge or hydrophobicity of the molecule at
the site of the substitution, and/or (c) the bulk of the side
chain.
[0057] In the present invention, an "LP polynucleotide" or a
"polynucleotide encoding an LP polypeptide" refers to a nucleic
acid molecule with a nucleotide sequence of an identified SEQ ID
number and its complementary sequence or "complement". It further
includes those polynucleotides of about equal length as the
molecule identified by the SEQ ID Number (the "reference molecule")
and capable of hybridizing, under stringent hybridization and wash
conditions, to polynucleotide sequences comprising the sequence
represented by the SEQ ID Number or the complement thereof. In
reference to a polynucleotide, the term "about equal length" means
the same number of total nucleotides as the reference molecule plus
or minus up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides, most
preferably plus or minus 0 nucleotides. The addition or deletion of
nucleotides may occur anywhere along the length of the
polynucleotide molecule and need not be contiguous although the
addition or deletion of nucleotides preferably occurs at the 5'
and/or 3' end(s) when compared to the reference molecule.
[0058] A polynucleotide or nucleic acid of the present invention
can be composed of any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. For example, polynucleotides can be composed
of single- and double-stranded DNA, DNA that is a mixture of
single- and double-stranded regions, single- and double-stranded
RNA, and RNA that is a mixture of single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded regions. A
polynucleotide may contain one or more modified nucleotides.
"Modified" bases include, for example, tritylated bases and unusual
bases such as inosine. A variety of such modifications can be made;
thus, "polynucleotide" embraces chemically, enzymatically, or
metabolically modified forms.
[0059] "LP variant" means an "active" polypeptide as defined below,
having at least about 90% amino acid sequence identity to a
reference LP polypeptide. Polypeptide variants include, for
instance, variations of LP121A, LP121B, LP121C and LP10034, wherein
one or more amino acid residues are added, substituted or deleted,
at the N- or C-terminus or within the sequences LP121A, LP121B,
LP121C and LP10034 (SEQ ID NOS: 8, 11, 12, and 14), not necessarily
contiguously. For example, LP10034 could be the reference
polypeptide and the polypeptide altered from the LP10034
polypeptide would be the LP polypeptide variant. Ordinarily, an LP
polypeptide variant will have at least about 90% amino acid
sequence identity, preferably at least about 91%, 92%, 93%, 94%,
95%, 96%, 97% sequence identity, more preferably at least about 98%
sequence identity, even more preferably at least about 99% amino
acid sequence identity with the amino acid sequence described
(i.e., the reference LP polypeptide), with or without the signal
peptide.
[0060] "Percent (%) amino acid sequence identity" with respect to
the LP amino acid sequences identified herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in a reference LP
polypeptide sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as ALIGN, ALIGN-2,
Megalign (DNASTAR) or BLAST (e.g., Blast, Blast-2, WU-Blast-2)
software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For example, the percent identity values used
herein can be generated using WU-BLAST-2 [Altschul et al., Methods
in Enzymology 266: 460-480 (1996)]. Most of the WU-BLAST-2 search
parameters are set to the default values. Those not set to default
values, (i.e., the adjustable parameters) are set with the
following values: overlap span=1; overlap fraction=0.125; word
threshold (T)=11; and scoring matrix=BLOSUM 62. For purposes
herein, a percent amino acid sequence identity value is determined
by dividing (a) the number of matching identical amino acid
residues between the amino acid sequence of the LP polypeptide of
interest and the comparison amino acid sequence of interest (i.e.,
the sequence against which the LP polypeptide of interest is being
compared) as determined by WU-BLAST-2, by (b) the total number of
amino acid residues of the LP polypeptide of interest,
respectively.
[0061] A "LP variant polynucleotide" or "LP variant nucleic acid
sequence" means a nucleic acid molecule encoding an active LP
polypeptide as defined below having at least 75% nucleic acid
sequence identity with an LP polynucleotide identified by a SEQ ID
NO. of the present invention. Ordinarily, an LP polypeptide will
have at least 75% nucleic acid sequence identity, more preferably
at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, even
more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%
nucleic acid sequence identity, yet more preferably at least 98%
nucleic acid sequence identity, even more preferably at least 99%
nucleic acid sequence identity with the nucleic acid sequence of
its corresponding nucleic acid represented by a SEQ ID NO. for the
reference LP polynucleotide.
[0062] "Percent (%) nucleic acid sequence identity" with respect to
the LP polynucleotide sequences identified herein is defined as the
percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in the reference LP sequence after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for
purposes of determining percent nucleic acid sequence identity can
be achieved in various ways that are within the skill in the art,
for instance, using publicly available computer software such as
ALIGN, Align-2, Megalign (DNASTAR), or BLAST (e.g., Blast, Blast-2)
software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For example, percent nucleic acid identity values
can be generated using the WU-BLAST-2 (BlastN module) program
(Altschul et al., Methods in Enzymology 266: 460-480 (1996)). Most
of the WU-BLAST-2 search parameters are set to the default values.
Those not set default values (i.e., the adjustable parameters), are
set with the following values: overlap span=1; overlap
fraction=0.125; word threshold (T)=11; and scoring matrix=BLOSUM62.
For purposes herein, a percent nucleic acid sequence identity value
is determined by dividing (a) the number of matching identical
nucleotides between the nucleic acid sequence of the
polypeptide-encoding nucleic acid molecule of interest and the
comparison nucleic acid molecule of interest (i.e., the sequence
against which the polypeptide-encoding nucleic acid molecule of
interest is being compared) as determined by WU-BLAST-2, by (b) the
total number of nucleotides of the polypeptide-encoding nucleic
acid molecule of interest.
[0063] In other embodiments, the LP variant polypeptides are
encoded by nucleic acid molecules that encode an active LP
polypeptide and which are capable of hybridizing, preferably under
stringent hybridization and wash conditions, to nucleotide
sequences encoding the full-length LP polypeptide of interest. This
scope of variant polynucleotides specifically excludes those
sequences that are known as of the filing and/or priority dates of
the present application.
[0064] The term "mature protein" or "mature polypeptide" as used
herein refers to the form(s) of the protein as would be produced by
expression in a mammalian cell. For example, it is generally
hypothesized that once export of a growing protein chain across the
rough endoplasmic reticulum has been initiated, proteins secreted
by mammalian cells have a signal peptide (SP) sequence which is
cleaved from the complete polypeptide to produce a "mature" form of
the protein. Oftentimes, cleavage of a secreted protein is not
uniform and may result in more than one species of mature protein.
The cleavage site of a secreted protein is determined by the
primary amino acid sequence of the complete protein and generally
cannot be predicted with complete accuracy. Methods for predicting
whether a protein has an SP sequence, as well as the cleavage point
for that sequence, are known in the art. A cleavage point may exist
within the N-terminal domain between amino acid 10 and amino acid
35. More specifically the cleavage point is likely to exist after
amino acid 15 but before amino acid 31. As one of ordinary skill
would appreciate, however, cleavage sites sometimes vary from
organism to organism and may even vary from molecule to molecule
within a cell and cannot be predicted with absolute certainty.
Optimally, cleavage sites for a secreted protein are determined
experimentally by amino-terminal sequencing of the one or more
species of mature proteins found within a purified preparation of
the protein.
[0065] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous.
[0066] The term "amino acid" is used herein in its broadest sense,
and includes naturally occurring amino acids as well as
non-naturally occurring amino acids, including amino acid analogs
and derivatives. The latter includes molecules containing an amino
acid moiety. One skilled in the art will recognize, in view of this
broad definition, that reference herein to an amino acid includes
naturally occurring proteogenic L-amino acids; D-amino acids;
chemically modified amino acids, such as amino acid analogs and
derivatives; naturally occurring non-proteogenic amino acids such
as norleucine, .beta.-alanine, ornithine, etc.; and chemically
synthesized compounds having properties known in the art to be
characteristic of amino acids. As used herein, the term
"proteogenic" indicates that the amino acid can be incorporated
into a peptide, polypeptide, or protein in a cell through a
metabolic pathway.
[0067] The terms "treating", "treatment" and "therapy" as used
herein refer to curative therapy, prophylactic therapy, and
preventive therapy. An example of "preventive therapy" is the
prevention or lessened targeted pathological condition or disorder.
Those in need of treatment include those already with the disorder
as well as those prone to have the disorder or those in whom the
disorder is to be prevented.
[0068] The term "agonist" as used herein refers to a molecule which
intensifies or mimics the biological activity of huJAM. Agonists
may include proteins, nucleic acids, carbohydrates, small
molecules, or any other compound or composition which modulates the
activity of huJAM either by directly interacting with huJAM or by
acting on component(s) of the biological pathway in which huJAM
participates.
[0069] The term "antagonist" refers to a molecule which inhibits or
attenuates the biological activity of huJAM. Antagonists may
include proteins, antibodies, nucleic acids, carbohydrates, small
molecules, or any other compound or composition which modulates the
activity of huJAM either by directly interacting with huJAM or by
acting on component(s) of the biological pathway in which huJAM
participates.
[0070] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption but, rather, is cyclic
in nature.
[0071] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0072] A "therapeutically-effective amount" is the minimal amount
of active agent (e.g., an LP polypeptide) which is necessary to
impart therapeutic benefit to a mammal. For example, a
"therapeutically-effectiv- e amount" to a mammal is such an amount
which induces, ameliorates or otherwise causes an improvement in
the pathological symptoms, disease progression, physiological
conditions associated with or resistance to succumbing to the
aforedescribed disorder.
[0073] "Carriers" as used herein include
pharmaceutically-acceptable carriers, excipients, or stabilizers
which are nontoxic to the cell or mammal being exposed thereto at
the dosages and concentrations employed. Often the
physiologically-acceptable carrier is an aqueous pH buffered
solution. Examples of physiologically acceptable carriers include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecule weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM., polyethylene glycol, and
PLURONIC.TM..
[0074] "Active" or "activity" in the context of variants of the LP
polypeptide refers to retention of biologic function of the
unmodified (or wild type) LP polypeptide and/or the ability to bind
to a receptor or ligand much as would an unmodified LP polypeptide
of the invention, and/or the ability to induce production of an
antibody against an antigenic epitope possessed by the LP
polypeptide at levels near that of the unmodified LP polypeptide.
More specifically, "biological activity" refers to a biological
function (either inhibitory or stimulatory) caused by a reference
LP polypeptide. Exemplary biological activities include, but are
not limited to, the ability of such molecules to induce or inhibit
infiltration of inflammatory cells (e.g., leukocytes) into a
tissue, to induce or inhibit adherence of a leukocyte to an
endothelial or epithelial cell, to stimulate or inhibit T-cell
proliferation or activation, to stimulate or inhibit cytokine
release by cells or to increase or decrease vascular
permeability.
[0075] Compositions and Methods of the Invention
[0076] The present invention is based in part upon the discovery
and synthesis of extracellular huJAM LP proteins (LP121A (SEQ ID
NO:11), LP121B (SEQ ID NO:12), LP121C (SEQ ID NO:8), LP10034 (SEQ
ID NO:14)) and their use in treating, preventing, and diagnosing
cancer, cardiovascular disorders, and immune system disorders such
as autoimmune diseases and inflammatory disorders.
[0077] 1. Preparation of Extracellular HuJAM LP Polypeptides
[0078] Nucleic acid encoding an LP polypeptide may be obtained from
a cDNA library prepared from tissue believed to possess the LP mRNA
and to express it at a detectable level. Libraries can be screened
with probes (such as antibodies to an LP polypeptide or
oligonucleotides of at least about 20-80 bases) designed to
identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic library with the selected probe may
be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, NY (1989). An alternative means to
isolate the gene encoding LP polypeptide is to use PCR methodology
(Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1995)).
Further details of the cloning and expression of LP121A and LP10034
are in the Examples herein.
[0079] The LP polypeptides of the present invention are
extracellular human junction adhesion molecules. These molecules
lack the transmembrane domain and cytoplasmic domain that are
present in the full-length, membrane-bound human junction adhesion
molecules. The LP polypeptides may have a signal peptide sequence
to enable protein transport within the cell; however, this signal
peptide sequence is not present in the mature huJAM polypeptides as
they exist when outside the cell.
[0080] A signal peptide, comprised of about 10-30 hydrophobic amino
acids, targets the nascent protein from the ribosome to the
endoplasmic reticulum (ER). Once localized to the ER, the proteins
can be further directed to the Golgi apparatus within the cell. The
Golgi distributes proteins to vesicles, lysosomes, the cell
membrane, and other organelles. Proteins targeted to the ER by a
signal sequence can be released from the cell into the
extracellular space. This is the case for the extracellular huJAM
polypeptides of the present invention. For example, vesicles
containing proteins to be moved outside the cell can fuse with the
cell membrane and release their contents into the extracellular
space via a process called exocytosis. Exocytosis can occur
constitutively or after receipt of a triggering signal. In the
latter case, the proteins are stored in secretory vesicles until
exocytosis is triggered. Proteins that transit through this pathway
are either released into the extracellular space or retained in the
plasma membrane. Protein that are retained in the plasma membrane
(e.g., full-length huJAM), contain one or more transmembrane
domains, each comprised of about 20 hydrophobic amino acid
residues. The LP polypeptides of the present invention lack both
the transmembrane domain and the downstream cytoplasmic domain of
the full-length huJAM.
[0081] The common structure of signal peptides from various
proteins is typically described as a positively charged n-region,
followed by a hydrophobic h-region and a neutral but polar
c-region. The (-3, -1) rule states that the residues at positions
-3 and -1 (relative to the signal peptide cleavage site) must be
small and neutral for cleavage to occur correctly.
[0082] In many instances the amino acids comprising the signal
peptide are cleaved off the protein during transport or once its
final destination has been reached. Specialized enzymes, termed
signal peptidases, are responsible for the removal of the signal
peptide sequences from proteins. These enzymes are activated once
the signal peptide has directed the protein to the desired
location.
[0083] LP polypeptides of interest may be produced recombinantly,
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, which optionally may be a signal sequence
or other polypeptide having a specific cleavage site at the
N-terminus of the mature protein or polypeptide. In general, the
signal sequence may be a component of an expression vector, or it
may be a part of the LP polypeptide-encoding DNA that is inserted
into such a vector. For E. coli expression, the signal sequence may
be a prokaryotic signal sequence selected, for example, from the
group of the alkaline phosphatase, penicillinase, lpp, or
heat-stable enterotoxin II leaders. For yeast secretion the signal
sequence may be, e.g., the yeast invertase leader, alpha factor
leader (including Saccharomyces and Kluyveromyces .alpha.-factor
leaders, the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP
362,179), or the signal described in WO 90/13646. In mammalian cell
expression, mammalian signal sequences may be used to direct
secretion of the protein, such as signal sequences from secreted
polypeptides of the same or related species as well as viral
secretory leaders.
[0084] Also provided by the present invention are nucleic acid
compositions encoding the LP polypeptides. These nucleic acids are
shown in SEQ ID NOS: 15 through 22 (FIGS. 11 through 16
respectively). Also provided are nucleic acids that are homologous
or substantially identical or stringently hybridize to the nucleic
acid shown in FIG. 11 through 16 as described herein.
[0085] 2. Extracellular HuJAM LP Polypeptide Variants
[0086] The present invention encompasses variants of a
polynucleotide sequence encoding an extracellular huJAM polypeptide
disclosed in SEQ ID NOS: 15 through 22 and their complementary
strands. The present invention also encompasses variants of the
extracellular huJAM polypeptide sequences disclosed in SEQ ID NOS:
7 through 14. The term "Variant" refers to a polynucleotide or
polypeptide differing from an LP polynucleotide sequence or an LP
polypeptide of the present invention, but retaining essential
properties thereof. Generally, variants are closely similar overall
in structural and/or sequence identity, and, in many regions,
identical to an LP polynucleotide or polypeptide of the present
invention. The term "variant" is further described in the
definitions herein.
[0087] The present invention encompasses nucleic acid molecules
that comprise as their sole source of extracellular huJAM sequence,
or alternatively consist of, a polynucleotide sequence that is at
least: 90%, 91%, 92%, 93%, 94% or more preferably at least 95%,
96%, 97%, 98%, or most preferably at least 99% identical to a
polynucleotide coding sequence of SEQ ID NOS: 15 through 22 (or a
strand complementary thereto); or a polynucleotide sequence
encoding a polypeptide of SEQ ID NO: 7 through 14.
[0088] Polynucleotides, that encode an extracellular huJAM and
stably hybridize to a nucleic acid molecule that comprises as its
sole source of extracellular huJAM sequence, or alternatively
consists of, a polynucleotide sequence that is at least: 90%, 91%,
92%, 93%, 94% or more preferably at least 95%, 96%, 97%, 98%, or
most preferably at least 99% identical to a polynucleotide coding
sequence of SEQ ID NOS: 15 through 22 (or a strand complementary
thereto); or a polynucleotide sequence encoding a polypeptide of
SEQ ID NOs: 7 through 14, under stringent hybridization and wash
conditions, are also encompassed by the invention, as are the
extracellular huJAM polypeptides encoded by these
polynucleotides.
[0089] The present invention is also directed to polypeptides that
comprise as their sole source of huJAM, or alternatively consist
of, an amino acid sequence that is at least: 90%, 95%, 96%, 97%,
98%, 99% identical to a polypeptide sequence of SEQ ID NOs: 7
through 14. A polynucleotide sequence having at least some
"percentage identity," (e.g., 95%) to another polynucleotide
sequence, means that the sequence being compared (e.g., the test
sequence or candidate sequence) may vary from another sequence
(e.g. the reference sequence) by a certain number of nucleotide
differences (e.g., a test sequence with 95% sequence identity to a
reference sequence can have, on average, up to five point mutations
per each 100 contiguous nucleotides of the referent sequence). In
other words, for a test sequence to exhibit at least 95% identity
to a reference sequence, up to 5% of the nucleotides in the
reference may differ, e.g., be deleted or substituted with another
nucleotide, or a number of nucleotides (up to 5% of the total
number of nucleotides in the reference sequence) may be inserted
into the reference sequence. As a practical matter, determining if
a particular nucleic acid molecule or polynucleotide sequence
exhibits at least about: 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity to an LP polynucleotide sequence can be
accomplished using known computer programs.
[0090] Typically, in such a sequence comparison, one sequence acts
as a reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequent coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percentage sequence identity for a test sequence(s) relative to the
reference sequence, based on the parameters of a designated
program.
[0091] Optimal alignment of sequences for comparison can be
conducted as described in the definitions herein or, e.g., by the
local homology algorithm of Smith and Waterman (1981) Adv. Appl.
Math. 2: 482, by the homology alignment algorithm of Needlman and
Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity
method of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:
2444, by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, Madison, Wis.), or by visual
inspection (see generally, Ausubel, et al. supra).
[0092] A typical method for determining, a best overall match (also
referred to as a global sequence alignment) between a test and a
referent sequence can be determined using, e.g., the FASTDB
computer program based on the algorithm of Brutlag, et al. (1990)
Comp. App. Biosci. 6: 237-245. In a FASTDB sequence alignment, the
test and reference sequences are, e.g., both DNA sequences. An RNA
sequence can be compared by converting U's to T's. The result of a
global sequence alignment is given in terms of a percentage
identity.
[0093] Typical parameters used in a FASTDB alignment of DNA
sequences to calculate percent identity are Matrix=Unitary,
k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization
Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty
0.05, Window Size=500, or the length of the referent nucleotide
sequence, whichever is shorter. If the reference sequence is
shorter than the test sequence because of 5' or 3' terminal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for 5' and 3' terminal
truncations of the subject sequence when calculating percent
identity. For reference sequences truncated at the 5' or 3'
terminal ends, relative to the test sequence, the percentage
identity is corrected by calculating the number of bases of the
test sequence that are 5' and 3' of the subject sequence, which are
not matched/aligned, as a percentage of the total bases of the test
sequence. Whether a nucleotide is matched/aligned is determined by
results of the FASTDB sequence alignment. This percentage is then
subtracted from the percentage identity, calculated by the above
FASTDB program using the specified parameters, to arrive at a final
percentage identity score. The corrected score is what is used for
the purposes of sequence identity for the present invention.
Ordinarily, bases outside the 5' and 3' bases of the subject
sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the test sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0094] For example, a 90 base reference sequence is aligned to a
100 base test sequence to determine percentage identity. The
deletions occur at the 5' end of the reference sequence and
therefore, the FASTDB alignment does not show a matched/alignment
of the first 10 bases at the 5' end. The 10 unpaired bases
represent 10% of the sequence (number of bases at the 5' and 3'
ends not matched/total number of bases in the test sequence) so 10%
is subtracted from the percentage identity score calculated by the
FASTDB program. If the remaining 90 bases were perfectly matched
the final percentage identity would be 90%.
[0095] In another example, a 90 base reference sequence is compared
with a 100 base test sequence. This time the deletions are internal
deletions so that there are no bases on the 5' or 3' of the subject
sequence, which are not matched/aligned with the test. In this
case, the percentage identity calculated by FASTDB is not manually
corrected. Again, only bases 5' and 3' of the subject sequence that
are not matched/aligned with the test sequence are manually
corrected for. No other manual corrections are made for the
purposes of the present invention.
[0096] Especially preferred are polynucleotide variants containing
alterations, which produce silent substitutions (i.e., no change in
amino acid encoded thereby), additions, or deletions, but do not
alter the properties or activities of the encoded polypeptide.
Nucleotide variants produced by silent substitutions due to the
degeneracy of the genetic code are preferred.
[0097] A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described herein. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described below.
[0098] A polypeptide exhibiting or having at least about, e.g., 95%
"sequence identity" to another amino acid sequence may include,
e.g., up to five amino acid alterations per each 100 amino acid (on
average) stretch of the test amino acid sequence. In other words, a
first amino acid sequence that is at least 95% identical to a
second amino acid sequence, can have up to 5% of its total number
of amino acid residues different from the second sequence, e.g., by
insertion, deletion, or substitution of an amino acid residue.
[0099] Alterations in amino residues of a polypeptide sequence may
occur, e.g., at the amino or carboxy terminal positions or anywhere
between these terminal positions, interspersed either individually
among residues in the sequence or in one or more contiguous
sections, portions, or fragments within the sequence.
[0100] As a practical matter, whether any particular polypeptide
sequence exhibits at least about: 90%, 91%, 92%, 93%, 94% 95%, 96%,
97%, 98%, or 99% similarity to another sequence, (e.g., SEQ ID Nos:
7 through 14) can be determined conventionally by using known
methods in the art, e.g., a computer algorithm such as
ClustalW.
[0101] A preferred method for determining the best overall match
(also called a global sequence alignment) between two sequences
(either nucleotide or amino acid sequences) uses the FASTDB
algorithm of Brutlag, et al. (1990) Comp. App. Biosci. 6: 237-245.
The result of such a global sequence alignment is given as a
percentage of sequence identity, e.g., with 100% representing
complete sequence identity.
[0102] Typical FASTDB parameters for amino acid alignments are,
e.g.,: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window
Size=sequence length, Gap Penalty=5 Gap Size Penalty=0.05, Window
Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[0103] If the subject sequence is shorter than the test sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N-and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the test sequence, the percent identity is corrected by
calculating the number of residues of the test sequence that are N-
and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the test sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percentage identity
score. This final percentage identity score is what is used for the
purposes of the present invention.
[0104] Only residues to the N- and C-termini of the subject
sequence, which are not matched/aligned with the test sequence, are
considered for the purposes of manually adjusting the percent
identity score. That is, only test residue positions outside the
farthest N- and C-terminal residues of the subject sequence. For
example, a 90 amino acid residue subject sequence is aligned with a
100-residue test sequence to determine percent identity. The
deletion occurs at the N-terminus of the subject sequence and
therefore, the FASTDB alignment does not show a matching/alignment
of the first 10 residues at the N-terminus. The unpaired residues
represent 10% of the sequence (number of residues at the N- and
C-termini not matched/total number of residues in the test
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 residues were
perfectly matched the final percent identity would be 90%.
[0105] In another example, a 90-residue subject sequence is
compared with a 100-residue test sequence. This time the deletions
are internal deletions so there are no residues at the N- or
C-termini of the subject sequence, which are not matched/aligned
with the test. In this case, the percent identity calculated by
FASTDB is not manually corrected. Once again, only residue
positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the test sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0106] Variants encompassed by the present invention may contain
alterations in the coding regions, non-coding regions, or both.
Moreover, variants in which 1-2, 1-5, or 5-10 amino acids are
substituted, deleted, or added in any combination are
preferred.
[0107] Variants may be produced by mutagenesis techniques or by
direct synthesis using known methods of protein engineering and
recombinant DNA technology. Such variants may be generated to
improve or alter the characteristics of an LP polypeptide or may
occur unintentionally. For instance, one or more amino acids can
often be deleted from the N-terminus or C-terminus of a secreted
polypeptide without a substantial loss of biological function.
[0108] Moreover, ample evidence demonstrates that polypeptide or
polynucleotide variants can retain a biological activity similar to
that of the naturally occurring protein. Moreover, even if deleting
one or more amino acids from the N-terminus or C-terminus of the
polypeptide results in modification or loss of one or more
biological functions, other biological activities may be
retained.
[0109] The invention also encompasses LP polypeptide variants that
show a biological activity of the reference huJAM such as, e.g.,
ligand binding or antigenicity. Such variants include, e.g.,
deletions, insertions, inversions, repeats, and substitutions
selected so as to have little effect on activity using general
rules known in the art. For example, teachings on making
phenotypically silent amino acid substitutions are provided, e.g.,
by Bowie, et al. (1990) Science 247: 1306-1310.
[0110] One technique compares amino acid sequences in different
species to identify the positions of conserved amino acid residues
since changes in an amino acid at these positions are more likely
to affect a protein function. In contrast, the positions of
residues where substitutions are more frequent generally indicates
that amino acid residues at these positions are less critical for a
protein function. Thus positions tolerating amino acid
substitutions typically may be modified while still maintaining a
biological activity of a protein.
[0111] Another technique uses genetic engineering to introduce
amino acid changes at specific positions of a polypeptide to
identify regions critical for a protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (the
introduction of single alanine mutations at every residue in the
molecule) can be used. (Cunningham and Wells (1989) Science 244:
1081-1085) A resulting mutant can subsequently be tested for a
biological activity.
[0112] These two techniques have revealed that proteins are
surprisingly tolerant of amino acid substitutions and they
generally indicate which amino acid changes are likely to be
permissive at certain amino acid positions in a protein. For
example, typically, most buried amino acid residues (those within
the tertiary structure of the protein) require nonpolar side
chains, whereas few features of surface side chains are generally
conserved. Preferred conservative amino acid substitutions are
listed in Table 1.
[0113] Besides using conservative amino acid substitutions, other
variants of the present invention include, but are not restricted
to (i) substitutions with one or more of the non-conserved amino
acid residues or (ii) substitution with one or more amino acid
residues having a substituent group, or (iii) fusion of the mature
polypeptide with another compound, such as a compound to increase
the stability and/or solubility of the polypeptide (e.g.,
polyethylene glycol), or (iv) fusion of the polypeptide with
additional amino acids, such as, e.g., an IgG Fc fusion region
peptide, or leader or secretory sequence, or a sequence
facilitating purification. All such variants would be within the
scope of those skilled in the art of molecular biology.
[0114] Polypeptide variants containing amino acid substitutions of
charged amino acids with other charged or neutral amino acids may
produce polypeptides with improved characteristics e.g., such as
less aggregation. Aggregation of pharmaceutical formulations both
reduces activity and increases clearance due to the aggregate's
immunogenic activity (Cleland, et al. (1993) Crit. Rev. Therapeutic
Drug Carrier Systems 10: 307-377).
[0115] A further embodiment of the invention encompasses a protein
that comprises an amino acid sequence of the present invention, as
the sole source of huJAM, that contains at least one amino acid
substitution, but not more than 20 amino acid substitutions,
preferably not more than 15 amino acid substitutions.
[0116] Of course, in order of ever-increasing preference, it is
highly preferable for an LP polypeptide of the invention to have an
amino acid sequence that comprises an amino acid sequence of the
present invention as the sole source of huJAM, which contains zero
or one, but not more than: 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino
acid substitutions; wherein conservative amino acid substitutions
are more preferable than non-conservative substitutions.
[0117] 3. Modifications of Extracellular HuJAM LP Polypeptides
[0118] LP polypeptides of the invention are composed of amino acids
joined to each other by peptide bonds or modified peptide bonds,
i.e., peptide isosteres, and may contain amino acids other than the
gene-encoded amino acids. The LP polypeptides may be modified by
either natural processes, such as posttranslational processing, or
by chemical modification techniques which are well known in the
art. Such modifications are well-described in the art.
Modifications can occur anywhere in the LP polypeptides, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. The same type of modification may be present in
the same or varying degrees at several sites in a given LP
polypeptide. Also, a given LP polypeptide may contain many types of
modifications. LP polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic LP polypeptides
may result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. See, for instance, Creighton, Proteins--Structure
and Molecular Properties, 2nd Ed., W.H. Freeman and Company, New
York (1993); Johnson, Posttransational Covalent Modification of
Proteins, Academic Press, New York, pp. 1-12 (1983); Seifter et
al., Meth. Enzymol. 182: 626-46 (1990); Rattan et al., Ann. NY
Acad. Sci. 663: 48-62 (1992).
[0119] A type of covalent modification of the LP polypeptides
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. "Altering the
native glycosylation pattern" is intended for purposes herein to
mean deleting one or more carbohydrate moieties found in native
sequence LP polypeptide and/or adding one or more glycosylation
sites that are not present in the native sequences of LP.
Additionally, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0120] Addition of glycosylation sites to LP polypeptides may be
accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequences of LP (for O-linked glycosylation sites). The LP
amino acid sequences may optionally be altered through changes at
the DNA level, particularly by mutating the DNA encoding the LP
polypeptides at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0121] Another means of increasing the number of carbohydrate
moieties on the LP polypeptides is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330, and in Aplin and
Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
[0122] Removal of carbohydrate moieties present on the LP
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art. Enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol. 138: 350-9 (1987).
[0123] Another type of covalent modification of LP polypeptides
comprises linking any one of the LP polypeptides to one of a
variety of nonproteinaceous 20 polymers, e.g., polyethylene glycol,
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
[0124] LP polypeptides of the present invention may also be
modified to form fusion molecules comprising an LP polypeptide as
the sole source of extracellular huJAM fused to a heterologous
polypeptide. In one embodiment, such a fusion molecule comprises a
fusion of an LP polypeptide with a tag polypeptide which provides
an epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of LP polypeptide. The presence of such epitope-tagged forms of LP
can be detected using an antibody against the tag polypeptide.
Also, provision of the epitope tag enables an LP polypeptide to be
readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag.
[0125] In an alternative embodiment, the fusion molecule may
comprise a fusion of an LP polypeptide which is the sole source of
huJAM with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of the fusion molecule, such a
fusion could be to the Fc region of an IgG molecule.
[0126] In yet a further embodiment, the LP polypeptides of the
present invention may also be modified in a way to form a fusion
molecule comprising an LP polypeptide of the invention which is the
sole source of huJAM fused to a leucine zipper at either the N- or
C-terminal end of the LP polypeptide. Various leucine zipper
polypeptides have been described in the art. It is believed that
use of a leucine zipper fused to an LP polypeptide may be desirable
to assist in dimerizing or trimerizing soluble LP polypeptides in
solution.
[0127] 4. Expression of LP Polypeptides
[0128] a. Expression Vector Construction
[0129] Recombinant expression vectors are typically
self-replicating DNA or RNA constructs containing a desired gene to
be expressed operably linked to a promoter and optionally other
control elements recognized in a suitable host cell. The specific
type of control elements necessary to effect expression depends on
the host cell used and the level of expression desired. Proteins
can be expressed in mammalian cells, yeast, bacteria, or other
cells under the control of appropriate promoters.
[0130] Vectors, as used herein, encompass plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles that
enable the integration of DNA fragments into the genome of the host
although, optionally, expression can occur transiently without
integration. Plasmids are the most commonly used form of vector,
but many other forms of vectors that perform an equivalent function
are also suitable for use (see, e.g., Pouwels, et al. (1985 and
Supplements) Cloning Vectors: A Laboratory Manual Elsevier, N.Y.;
and Rodriquez, et al. (eds.) (1988) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses Buttersworth, Boston, Mass.).
[0131] Both expression vectors and cloning vectors contain a
nucleic acid sequence that enables the vector to replicate in one
or more selected host cells. Such sequences are well known for a
variety of bacteria, yeast, and viruses.
[0132] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement autotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0133] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the LP polypeptide-encodingnucleic acid, such as DHFR or
thymidine kinase. An appropriate host cell when wild-type DHFR is
employed is the CHO cell line deficient in DHFR activity, prepared
and propagated as described Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA, 77: 4216-20 (1980). A suitable selection gene for use in
yeast is the trpl gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature 282: 39-43 (1979); Kingsman et al., Gene
7: 141-52 (1979); Tschumper et al., Gene 10: 157-66 (1980)]. The
trpl gene provides a selection marker for a mutant strain of yeast
lacking the ability to grow in tryptophan, for example, ATCC No.
44076 or PEPC1 [Jones, Genetics 85: 23-33 (1977)].
[0134] Expression vectors contain a promoter operably linked to the
LP polypeptide-encoding nucleic acid sequence to direct mRNA
synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters for use in bacterial systems also
will contain a Shine-Dalgarno (S.D.) sequence operably linked to
the DNA encoding an LP polypeptide of interest.
[0135] Transcription of a DNA encoding an LP polypeptide by higher
eukaryotes may be increased by inserting an enhancer sequence into
the vector. Enhancers are cis-acting elements of DNA, usually about
from 10 to 300 bp, that act on a promoter to increase its
transcription level. Many enhancer sequences are now known from
mammalian genes (globin, elastase, albumin, a-ketoprotein, and
insulin). Typically, however, one will use an enhancer from a
eukaryotic-cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
The enhancer may be spliced into the vector at a position 5' or 3'
to the LP polypeptide coding sequence.
[0136] Expression vectors used in eukaryotic host cells will also
contain sequences necessary for the termination of transcription
and optionally for stabilizing the mRNA. Such sequences are
commonly available from the 5' and occasionally 3' untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an LP polypeptide.
[0137] b. Expression in Host Cells
[0138] The description below relates primarily to production of LP
polypeptide by culturing cells transformed or transfected with a
vector containing LP polypeptide-encoding nucleic acid. It is, of
course, contemplated that alternative methods, which are well known
in the art, may be employed to prepare LP polypeptides. For
instance, the LP sequence, or portions thereof, may be produced by
direct peptide synthesis using solid-phase techniques [see, e.g.,
Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co.,
San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85:
2149-2154 (1963)]. In vitro protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be accomplished, for instance, using an Applied Biosystems Peptide
Synthesizer (Foster City, Calif.) using manufacturer's
instructions. Various portions of a LP polypeptide may be
chemically synthesized separately and combined using chemical or
enzymatic methods to produce a full-length LP.
[0139] Host cells are transfected or transformed with expression
vectors or cloning vectors described herein for LP polypeptide
production and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. The culture
conditions, such as media, temperature, pH and the like, can be
selected by the skilled artisan without undue experimentation. In
general, principles, protocols, and practical techniques for
maximizing the productivity of cell cultures can be found in
Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.
(IRL Press, 1991) and Sambrook et al., supra. Methods of
transfection are known to the ordinarily skilled artisan, for
example, CaPO.sub.4 and electroporation.
[0140] Suitable host cells for cloning or expressing the nucleic
acid (e.g., DNA) in the vectors herein include prokaryote, yeast,
or higher eukaryote cells. Suitable prokaryotes include but are not
limited to E. coli K12 strain MM294 (ATCC 3 1.446); E. coli Xl 776
(ATCC 3 1.537); E. coli strain W3110 (ATCC 27.325) and KS 772 (ATCC
53.635). Other suitable prokaryotic host cells include
Enterobacter, Erwinia, Klebisella, Proteus, Salmonella, Serratia,
and Shigeila, as well as Bacilli, Pseudomona, and Streptomyces.
These examples are illustrative rather than limiting. Strain W3110
is one particularly preferred host or parent host because it is a
common host strain for recombinant DNA product fermentations.
Preferably, the host cell secretes minimal amounts of proteolytic
enzymes. Alternatively, in vivo methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0141] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for LP polypeptide expressing vectors. Saccharomyces cerevisiae is
a commonly used lower eukaryotic host microorganism. Many others
are used by those in the art.
[0142] Suitable host cells for the expression of glycosylated LP
polypeptides of the invention are derived from multicellular
organisms. Examples of invertebrate cells include insect cells such
as Drosophila S2 and Spodoptera Sp, Spodoptera highs as well as
plant cells. Examples of useful mammalian host cell lines include
Chinese hamster ovary (CHO) and COS cells. Additional examples
include the monkey kidney CVl line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line [293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol., 36:
59-74 (1977)]; Chinese hamster ovary cells/-DHFR [CHO, Urlaub and
Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-20 (1980)]; mouse
sertoli cells [TM4, Mather, Biol. Reprod. 23: 243-52 (1980)]; human
lung cells (W138. ATCC CCL 75); human liver cells (Hep G2, HB
8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The
selection of the appropriate host cell is within the skill in the
art.
[0143] c. LP Polypeptide Purification
[0144] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA 77: 5201-5 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0145] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence provided herein or against exogenous sequence
fused to an LP polypeptide-encoding DNA and encoding a specific
antibody epitope.
[0146] Forms of LP polypeptides may be recovered from culture
medium or from host cell lysates. The LP polypeptides of the
present invention are not membrane-bound. Cells employed in
expression of LP polypeptides can be disrupted by various physical
or chemical means, such as freeze-thaw cycling, sonication,
mechanical disruption, or cell lysing agents.
[0147] It may be desirable to purify LP polypeptides away from
another recombinant cell polypeptide. The following procedures are
exemplary of suitable purification procedures: fractionation on an
ion-exchange column; ethanol precipitation; reversed-phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; protein A
Sepharose columns to remove contaminants such as IgG; and metal
chelating columns to bind epitope-tagged forms of LP polypeptides.
Various methods of protein purification may be employed and such
methods are known in the art and described, for example, in
Deutscher, Methods in Enzymology 182: 83-9 (1990) and Scopes,
Protein Purification: Principles and Practice, Springer-Verlag, NY
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular LP
polypeptide produced. Purification of the LP polypeptides of the
invention are futher described in the Examples herein.
[0148] 5. LP Polypeptide Analysis
[0149] Many types of analyses can be performed with the LP
polypeptides of the present invention to demonstrate their role in
the development, pathogenesis, and treatment of cancer,
cardiovascular disease and immune related disease, e.g.,
inflammation. Certain analyses are exemplified in the Examples
herein. Protocols for the analyses may be found in Sambrook, et
al., supra and other protocol texts used routinely in the art.
[0150] The location of tissues expressing the LP polypeptides can
be identified by determining mRNA expression in various human
tissues. Such a measurement can be made, for example, by Northern
blotting, dot blotting, or in situ hybridization based on the
sequences provided herein. Alternatively, antibodies may be used
that recognize specific duplexes. The location of a gene in a
specific tissue can be performed, for example, by Southern
blotting.
[0151] Cell-based assays using a cell type (optionally known to be
involved in a particular disease) are transfected with a vector
expressing an LP polypeptide of the invention. Such cells are
monitored for phenotypic changes, for example T-cell proliferation
by mixed lymphocyte reaction, inflammatory cell infiltration,
cytokine levels, JAM expression level variation, ligand binding and
reaction to particular antibodies. While transiently-transfected
cells can be used, stable cell lines expressing extracellular huJAM
are preferred.
[0152] Animal models can be used to further understand the role of
the LP polypeptides of the invention as demonstrated, for example,
in the Examples herein. Example 7 herein demonstrates that the LP
polypeptides of the invention may be used for blocking homotypic
and/or heterotypic JAM signaling or JAM interactions and therefore
can be useful for the prevention, treatment and diagnosis of
cardiovascular disease as overexpression of full-length huJAM lead
to an enlarged heart phenotype in a Xenopus embryo model. Example 9
herein demonstrates that the LP polypeptides of the invention
prevent LPS induced mortality in mice and therefore are useful as
an anti-inflammatory agent.
[0153] 6. Pharmaceutical Compositions
[0154] When the coding sequence for an LP polypeptide encodes a
protein which binds to another protein as is the case for
extracellular huJAM polypeptides of the present invention, the LP
polypeptide can be used in assays to identify the other proteins or
molecules involved in the binding interaction. By such methods,
inhibitors of the receptor/ligand binding interaction can be
identified. Proteins involved in such binding interactions can also
be used to screen for peptide or small molecule inhibitors or
agonists of the binding interaction. Also, the receptor LP
polypeptide can be used to isolate correlative ligand(s). Screening
assays can be designed to find lead compounds that mimic the
biological activity of a native LP polypeptide of the invention or
a receptor for an LP polypeptide of the invention. Such screening
assays will include assays amenable to high-throughput screening of
chemical libraries, making them particularly suitable for
identifying small molecule drug candidates. Small molecules
contemplated include synthetic organic or inorganic compounds. The
assays can be performed in a variety of formats, including
protein-protein binding assays, biochemical screening assays,
immunoassays and cell based assays, which are well characterized in
the art.
[0155] The LP polypeptides of the present invention may also be
administered via gene therapy protocols. "Gene therapy" includes
both conventional gene therapy, where a lasting effect is achieved
by a single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA.
[0156] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cell in vitro
or in vivo in the cells of the intended host. Techniques suitable
for the transfer of nucleic acid into mammalian cells in vitro
include the use of liposomes, electroporation, microinjection, cell
fusion, DEAE-dextran, the calcium phosphate precipitation method,
etc. The currently preferred in vivo gene transfer techniques
include transfection with viral (typically, retroviral) vectors and
viral coat protein-liposome mediated transfection [Dzau et al.,
Trends in Biotechnology 11: 205-10 (1993)]. In some situations it
is desirable to provide the nucleic acid with an agent that targets
the target cells, such as an antibody specific for a cell surface
membrane protein or the target cell or a ligand for a receptor on
the target cells. Where liposomes are employed, proteins which bind
to a cell surface membrane protein associated with endocytosis may
by used for targeting and/or to facilitate uptake, e.g., capsid
proteins or fragments thereof trophic for a particular cell type,
antibodies for proteins which undergo internalization in cycling,
proteins that target intracellular localization and enhance
intracellular half-life. The technique of receptor-mediated
endocytosis is described, for example by Wu et al., J. Biol. Chem.
262: 4429-32 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA
87: 3410-4 (1990). For a review of gene marking and gene therapy
protocols, see Anderson, Science 256: 808-13 (1992).
[0157] 7. Methods of Treatment
[0158] LP121A, LP121B, LP121C, LP10034 and variants thereof are
useful for the prevention, diagnosis, and treatment of cancer,
cardiovascular disorders and immune system disorders such as
autoimmune diseases and inflammatory disorders.
[0159] Data presented within the Examples herein demonstrate that
overexpression of the full-length, membrane-bound huJAM in the
heart leads to an enlarged heart phenotype. Further histological
anaysis demonstrated that the heart was dilated. It is contemplated
from these data that overexpression of full-length huJAM is
associated with conditions of human heart disease and that blocking
JAM signaling and/or interactions can be useful for preventing,
reversing or improving upon conditions of cardiovascular disease.
One method of blocking JAM signaling and homotypic and/or
heterotypic interactions would be by overexpression of
extracellular huJAM(s).
[0160] Further data presented in the Examples herein demonstrate
that injection of LPS into mice induces a major inflammatory
reaction that, with the dose used, is lethal. Substances that have
the ability to reverse this effect have anti-inflammatory
properties. DNA encoding extracellular huJAM, when injected into
the mice, prevented the LPS-induced mortality demonstrating that
huJAM acts as an anti-inflammatory agent.
[0161] Particular cancers suitable for treatment with the LP
polypeptides of the invention include, but are not limited to,
acute myelogenous leukemias including acute monocytic leukemia,
acute myeloblastic leukemia, acute promyelocytic leukemia, acute
myelomonocytic leukemia, acute erythroleukemia, acute
megakaryocyticleukemia, and acute undifferentiated leukemia, etc.;
and chronic myelogenous leukemias including chronic myelomonocytic
leukemia and chronic granulocyticleukemia. Additional cancers
suitable for treatment with the LP polypeptides of the invention
inculde, but are not limited to, adenocarcinoma, lymphoma,
melanoma, myeloma, Hamartoma, sarcoma, teratocarcinoma, and, in
particular, a cancer of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus.
[0162] Particular cardiovascular disorder suitable for treatment
with the LP polypeptides of the invention include, but are not
limited to, congestive heart failure, ischemic heart disease,
angina pectoris, myocardial infarction, hypertensive heart disease,
degenerative valvular heart disease, calcific aortic valve
stenosis, congenitally bicuspid aortic valve, mitral annular
calcification, mitral valve prolapse, rheumatic fever and rheumatic
heart disease, infective endocarditis, nonbacterial thrombotic
endocarditis, endocarditis of systemic lupus erythematosus,
carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis,
neoplastic heart disease, congenital heart disease, complications
of cardiac transplantation, arteriovenous fistula, atherosclerosis,
hypertension, vasculitis, Raynaud's disease, aneurysms, arterial
dissections, varicose veins, thrombophlebitis and phlebothrombosis,
vascular tumors, and complications of thrombolysis, balloon
angioplasty, vascular replacement, and coronary artery bypass graft
surgery.
[0163] Particular immune system disorders suitable for treatment
with the LP polypeptides of the invention include, but are not
limited to, inflammatory disorders, acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma.
[0164] Therapeutic formulations are prepared for storage by mixing
the active ingredient having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers [Remington's Pharmaceutical Sciences 16th edition
(1980)], in the form of lyophilized formulations or aqueous
solutions.
[0165] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0166] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition (1980).
[0167] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0168] Therapeutic compositions herein generally are placed into a
container having a sterile access port, for example, and
intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
[0169] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the therapeutic
agent(s), which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels [for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)],
polylactides, copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. Microencapsulation of recombinant
proteins for sustained release has been successfully performed with
human growth hormone (rhGH), interferon, and interleukin-2. Johnson
et al., Nat. Med. 2: 795-9 (1996); Yasuda et al., Biomed. Ther. 27:
1221-3 (1993); Hora et al., BioTechnology 8: 755-8 (1990); Cleland,
"Design and Production of Single Immunization Vaccines Using
Polylactide Polyglycolide Microsphere Systems" in Vaccine Design:
The Subunit and Adjuvant Approach, Powell and Newman, Eds., Plenum
Press, NY, 1995, pp. 439-462 WO 97/03692; WO 96/40072; WO 96/07399;
and U.S. Pat. No. 5,654,010.
[0170] The sustained-release formulations of these proteins may be
developed using polylactic-coglycolic acid (PLGA) polymer due to
its biocompatibility and wide range of biodegradable properties.
The degradation products of PLGA, lactic and glycolic acids, can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on
its molecular weight and composition. See Lewis, "Controlled
release of bioactive agents from lactide/glycolide polymer" in
Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker; New
York, 1990), M. Chasin and R. Langer (Eds.) pp. 1-41.
[0171] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated antibodies remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity.
[0172] The active agents of the present invention are administered
to a mammal, preferably a human, in accord with known methods, such
as intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerebral, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, intraoccular, intralesional, oral,
topical, inhalation, pulmonary, and/or through sustained
release.
[0173] Other therapeutic regimens may be combined with the
administration of an LP polypeptide of the invention.
[0174] For the prevention or treatment of disease, the appropriate
dosage of an active agent will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
whether the agent is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the agent, and the discretion of the attending
physician. The agent is suitably administered to the patient at one
time or over a series of treatments.
[0175] Dosages and desired drug concentration of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary artisan. Animal experiments provide reliable guidance for
the determination of effective does for human therapy. Interspecies
scaling of effective doses can be performed following the
principles laid down by Mordenti and Chappell, "The Use of
Interspecies Scaling in Toxicokinetics," in Toxicokinetics and New
Drug Development, Yacobi et al., Eds., Pergamon Press, NY 1989, pp.
4246.
[0176] When in vivo administration of an LP polypeptide of the
invention is employed, normal dosage amounts may vary from about 1
ng/kg up to 100 mg/kg of mammal body weight or more per day,
preferably about 1 pg/kg/day up to 100 mg/kg of mammal body weight
or more per day, depending upon the route of administration.
Guidance as to particular dosages and methods of delivery is
provided in the literature; see, for example, U.S. Pat. Nos.
4,657,760, 5,206,344 or 5,225,212. It is within the scope of the
invention that different formulations will be effective for
different treatment compounds and different disorders, that
administration targeting one organ or tissue, for example, may
necessitate delivery in a manner different from that to another
organ or tissue. Moreover, dosages may be administered by one or
more separate administrations or by continuous infusion. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. However, other dosage
regimens may be useful. The progress of this therapy is readily
monitored by conventional techniques and assays.
[0177] 8. Article of Manufacture
[0178] In another embodiment of the invention, an article of
manufacture containing materials useful for the diagnosis or
treatment of the disorders described above is provided. The article
of manufacture comprises a container and a label. Suitable
containers include, for example, bottles, vials, syringes, and test
tubes. The containers may be formed from a variety of materials
such as glass or plastic. The container holds a composition which
is effective for diagnosing or treating the condition and may have
a sterile access port (for example, the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). The active agent in the composition
is typically an LP polypeptide of the invention. The label on, or
associated with, the container indicates that the composition is
used for diagnosing or treating the condition of choice. The
article of manufacture may further comprise a second container
comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including other buffers, diluents, filters,
needles, syringes, and package inserts with instructions for
use.
[0179] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and not by way of
limitation.
2TABLE 2 SEQ ID LP# Name Type FIG. 1 full-length HuJAM1 amino acid
2B 2 full-length HuJAM2 amino acid 3B 3 full-length HuJAM3 amino
acid 4B 4 full-length HuJAM1 nucleotide 2A 5 full-length HuJAM2
nucleotide 3A 6 full-length HuJAM3 nucleotide 4A 7 HuJAM1 (aa.sup.+
1-235) amino acid 5 8 LP121C HuJAM1 (aa 28-235) amino acid 6 9
HuJAM2 (aa 1-224) amino acid 7a 10 HuJAM2 (aa 1-236) amino acid 7b
11 LP121A HuJAM2 (aa 29-224) amino acid 8a 12 LP121B HuJAM2 (aa
29-236) amino acid 8b 13 HuJAM3 (aa 1-240) amino acid 9 14 LP10034
HuJAM3 (aa 31-240) amino acid 10 15 encodes LP121C* HuJAM1(aa
1-235) nucleotide 11 16 encodes LP121A* HuJAM2(aa 1-224) nucleotide
12A 17 encodes LP121B* HuJAM2(aa 1-236) nucleotide 12B 18 encodes
LP10034* HuJAM3(aa 1-240) nucleotide 13 19 encodes LP121C*
HuJAM1(aa 28-235) nucleotide 14 20 encodes LP121A* HuJAM2(aa
29-224) nucleotide 15A 21 encodes LP121B* HuJAM2(aa 29-236)
nucleotide 15B 22 encodes LP10034* HuJAM3(aa 31-240) nucleotide 16
.sup.+aa means amino acid *SEQ ID NOS 15, 16, 17, and 18 encode the
extracellular huJAM and the signal peptide; SEQ ID NOS 19, 20, 21,
22 encode the extracellular huJAM without the signal peptide.
EXAMPLES
[0180] General Methods
[0181] Commercially available reagents referred to in the examples
are used according to manufacturer's instructions unless otherwise
indicated. The source of cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Unless otherwise noted, the present invention uses standard
procedures of recombinant DNA technology such as those described or
referenced in Sambrook, et al., (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, NY; Ausubel, et al.
(1989 and supplements) Current Protocols in Molecular Biology,
Green Publishing Associates and Wiley Interscience, NY; Innis et
al., or (1990) PCR Protocols: A Guide to Methods and Applications,
Academic Press, Inc., N.Y. Unless otherwise noted, the present
invention uses standard procedures of protein purification such as
those described or referenced in Methods in Enzymology vol. 182 and
other volumes in this series; Coligan, et al. (1995 and
supplements) Current Protocols in Protein Science, John Wiley and
Sons, New York, N.Y.; A Practical Guide to Protein and Peptide
Purification for Microsequencing, Academic Press, San Diego,
Calif.; or manufacturer's literature on use of protein purification
products, e.g., Pharmacia (Piscataway, N.J.) and Bio-Rad (Richmond,
Calif.).
Example 1
Cloning of LP121A, LP121B, LP121C and LP10034 into a Mammalian
Expression Vector
[0182] Flag-HIS (FLIS)-tagged version of human LP121A (SEQ ID
NO:11), LP121B (SEQ ID NO:12), LP121C (SEQ ID NO:8) and LP10034
(SEQ ID NO:14) are expressed in mammalian cells (e.g., HEK-293EBNA,
Cos-7 (ATCC CRL-1651) or HEK-293T (ATCC) in order to generate
enough recombinant protein for study. For example, the human LP121A
cDNA (SEQ ID NO. 16) is engineered for expression as follows. The
pINCY vector containing an Asc I/EcoR V fragment encoding the
full-length human JAM2 is used as a template to PCR amplify the
coding region of the cDNA.
[0183] The pINCY vector (Incyte Genomics, Palo Alto, Calif.) is a
derivative of pSPORT1 vector (Invitrogen, Carlsbad, Calif.) created
by removing the EcoRI restriction enzyme site by Klenow-filling and
religating; removing the Hind III restriction enzyme site by Klenow
filling and then inserting into this site a 10-mer EcoRI linker
(5'-CGGAATTCCG-3').
[0184] For LP121A, the PCR oligonucleotide primers are
5'-TTACTAGGCTGGCGCGCCACCATGGCGAGGAGGAGC-3' (SEQ ID NO: 23)
containing Asc I endonuclease restriction site (underlined) and
5'-AGGAAGGAATGCGCAAATTAT- AAAGGATTTTGTGT-3' (SEQ ID NO: 24)
containing an Fsp I restriction site (underlined) for the forward
and reverse strands, respectively. The resultant 702-bp PCR
generated fragment is cleaved with Asc I and Fsp I, gel-purified
and ligated into the mammalian expression vector pEW1969 (a
derivative of pJB02, Eli Lilly) that is digested with Asc I and
EcoR V to ultimately create vector pEW1969LP121A. Alternative
mamalian expression vectors can be used in place of pEW1969. Such
vectors are available from multiple companies including Promega
Corp. Madison, Wis. and Clontech, Palo Alto, Calif. This construct
is designed to express an extracellular huJAM2 molecule spanning
amino acids 1-224 of the full-length huJAM2 (including the
NH2-terminal amino acids which constitute the signal peptide) and
the FLAG/HIS tag at the COOH-terminus of the protein. The
expression is under the control of the CMV promoter. The final
construct is also designed to produce RNA that is functional in
Xenopus embryos in order to study overexpression phenotypes of the
protein in Xenopus embryos. One of skill in the art knows how to
adapt this method, or alternative methods cited in references
hereinabove, for construction of mammalian expression vectors
containing SEQ ID NOS:15 through 22.
[0185] As an additional cloning example, the human LP10034 cDNA
(SEQ ID NO: 18) is engineered for expression as follows. A pINCY
vector containing a fragment encoding the full-length of human
LP10034 (Incyte Genomics) is used as a template to PCR amplify,
using standard PCR conditions, the coding region of the cDNA. The
oligonucleotide primers are
(5'gatcggcgcgccagccaccATGGCGCTGAGGCGGCCA-3' (SEQ ID NO: 25))
containing an Asc I endonuclease restriction site (underlined) and
(5' cgccggtttaaacgcCAGGTCATAGACTTCCAT-3' (SEQ ID NO: 26))
containing a Pme I restriction site (underlined) for the forward
and reverse strands, respectively. The resultant 720-bp PCR
generated fragment is cleaved with Asc I and Pme I, gel-purified
and ligated into a mammalian expression vector, e.g., pPR1 (a
derivative of pJB02, Eli Lilly) that is digested with Asc I and Pme
I to ultimately create vector pPR1LP10034. This construct is
designed to express a truncated molecule (including the
NH2-terminal amino acids, which constitute the signal peptide) that
is extracellular targeted and the tag at the COOH-terminus of the
protein (total of 259 amino acid residues). The expression is under
the control of the CMV promoter.
[0186] HT-FlagHIS Protein Production
[0187] The vectors are separately transiently transfected into
mammalian cells (e.g., HEK-293, COS-7 or HEK-293T (Pear, W. S. et
al. (1993) PNAS 90: 8392-8396) using FUGENE as described by the
vendor (Roche). The recombinant proteins are measured in
supernatants collected from the transfected cells by Western-blot
using anti-Flag antibody and Coomassie-stained analyses.
Example 2
Expression and Purification of LP Polypeptides in E. coli
[0188] The bacterial expression vector pQE60 is used for bacterial
expression in this example although other bacterial expression
vectors are commercially available (QIAGEN, Inc., Chatsworth,
Calif.). pQE60 encodes an ampicillin antibiotic resistance gene
(Amp.sup.r) and contains a bacterial origin of replication (ori),
an IPTG-inducible promoter, a ribosome binding site (RBS), six
codons encoding histidine (His6 tag) residues that allow affinity
purification using nickel-nitrilo-tri-acetic acid (Ni-NTA) affinity
resin sold by QIAGEN, Inc., and single restriction enzyme cleavage
sites suitable for cloning (i.e., in a multiple cloning site).
These elements are arranged such that a DNA fragment encoding a
polypeptide of interest can be operably linked in such a way as to
produce that polypeptide with the six His residues covalently
linked to the carboxyl terminus of that polypeptide. However, a
polypeptide coding sequence can optionally be inserted in such a
way that translation of the six His codons is prevented and,
therefore, a polypeptide is produced with no 6.times.His tag.
[0189] The nucleic acid sequence encoding the desired portion of
LP121A, LP121B, LP121C or LP10034 lacking the hydrophobic leader
sequence is amplified from a cDNA clone using PCR oligonucleotide
primers, which anneal, one upstream (or 5') and one downstream (or
3'), to the desired portion of the LP polypeptide. Exemplary
primers for LP121A and LP10034 are described in Example 1 supra.
Additional nucleotides containing restriction sites to facilitate
cloning in the pQE60 vector may be added to the 5' and 3'
sequences, respectively.
[0190] The amplified nucleic acid fragments (e.g., those described
in Example 1 hereinabove) and the vector pQE60 are digested with
appropriate restriction enzymes and the digested DNAs are then
ligated together. Insertion of the LP polypeptide DNA into the
restricted pQE60 vector places the LP polypeptide coding region
including its associated stop codon downstream from the
IPTG-inducible promoter and operably linked in-frame with an
initiating AUG codon. The associated stop codon prevents
translation of the six histidine codons downstream of the insertion
point.
[0191] The ligation mixture is transformed into competent E. coli
cells using standard procedures. E. coli strain M15/rep4,
containing multiple copies of the plasmid pREP4, which expresses
the lac repressor and confers kanamycin resistance (Kan.sup.r), is
used in carrying out the illustrative example described herein.
This strain, which is only one of many that are suitable for
expressing polypeptides, is available commercially from QIAGEN,
Inc. Transformants are identified by their ability to grow on LB
plates in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis, PCR and DNA sequencing.
[0192] Bacteria containing the desired cloned constructs are grown
overnight (O/N) in liquid culture in LB media (Sigma Corp. St.
Louis, Mo.) supplemented with both ampicillin (100 .mu.g/ml) and
kanamycin (25 .mu.g/ml). The O/N culture is used to inoculate a
large culture, at a dilution of approximately 0.1:25 to 1:250. The
cells are grown to an optical density at 600 nm (OD600) of between
0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside (IPTG) is then
added to a final concentration of 1 mM to induce transcription from
the lac repressor sensitive promoter, by inactivating the lacI
repressor. Cells subsequently are incubated further for 3 to 4
hours. Cells then are harvested by centrifugation.
[0193] The cells are then stirred for 3-4 hours at 4.degree. C. in
6 M guanidine-HCl, pH 8.0. The cell debris is removed by
centrifugation, and the supernatant containing the LP polypeptide
is dialyzed against 50 mM Na-acetate buffer pH 6.0, supplemented
with 200 mM NaCl. Alternatively, a polypeptide can be successfully
refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM
Tris/HCl pH 7.4, containing protease inhibitors.
[0194] If insoluble protein is generated, the protein is made
soluble according to known method steps. After renaturation, the
polypeptide is purified by ion exchange, hydrophobic interaction,
and size exclusion chromatography. Alternatively, an affinity
chromatography step such as an antibody column is used to obtain
purified LP polypeptide. The purified polypeptide is stored at
4.degree. C. or frozen at -40.degree. C. to -120.degree. C.
Example 3
Cloning and Expression of LP Polypeptides in a Baculovirus
Expression System
[0195] In this example, the plasmid shuttle vector pA2 GP is used
to insert the cloned DNA encoding the LP polypeptide, without the
sequence encoding the signal peptide, into a baculovirus to express
the LP polypeptide, using a baculovirus leader and standard methods
as described in Summers, et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station Bulletin No. 1555 (1987). This
exemplary baculovirus expression vector contains the strong
polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the secretory signal
peptide (leader) of the baculovirus gp67 polypeptide and convenient
restriction sites such as BamH I, Xba I, and Asp 718. The
polyadenylation site of the simian virus 40 (SV40) is used for
efficient polyadenylation. For easy selection of recombinant virus,
the plasmid contains the beta-galactosidase gene from E. coli under
control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA to
generate viable virus that expresses the cloned polynucleotide.
[0196] Other baculovirus vectors can be used in place of the vector
above, e.g., pAc373, pVL941 and pAcIM1, as one skilled in the art
would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an
operably-linked AUG start codon as required. Such vectors are
described, for instance, in Luckow, et al., Virology 170:
31-39.
[0197] The cDNA sequence encoding the mature LP polypeptide of
interest lacking the AUG initiation codon (e.g., SEQ ID Nos 19, 20,
21, and 22) and the naturally associated nucleotide binding site,
is amplified using PCR oligonucleotide primers corresponding to
sequences upstream (5') and downstream (3') of the polynucleotide
encoding the LP polypeptide of interest. Non-limiting examples
include 5' and 3' primers having nucleotides corresponding to, or
complementary to, a portion of the coding sequence of a LP
polypeptide, according to method steps known to those of skill in
the art.
[0198] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit (e.g., GENECLEAN, Qbiogene,
Carlsbad, Calif.). The fragment is then digested with the
appropriate restriction enzyme and again is purified on a 1%
agarose gel. This fragment is designated herein "F1".
[0199] The plasmid is digested with the corresponding restriction
enzymes and optionally, can be dephosphorylated using calf
intestinal alkaline phosphatase, using routine procedures known in
the art. The DNA is then isolated from a 1% agarose gel using a
commercially available kit. This vector DNA is designated herein
"V1".
[0200] Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts (e.g., XL-1 Blue, Stratagene, La Jolla, Calif.) cells
are transformed with the ligation mixture and spread on culture
plates. Bacteria are identified that contain the plasmid bearing
the polynucleotide encoding the LP polypeptide of interest using
the PCR method, in which one of the primers is that used to amplify
the nucleic acid and a second primer is from well within the vector
so that only those bacterial colonies containing the nucleic acid
fragment encoding the LP polypeptide of interest will amplify the
DNA. The sequence of the cloned fragment is confirmed by DNA
sequencing. This plasmid is designated herein as pBacLP.
[0201] Five micrograms of the pBacLP plasmid is co-transfected with
1.0 .mu.g of a commercially available linearized baculovirus DNA
(BACULOGOLD.TM. baculovirus DNA, Pharmingen, San Diego, Calif.),
using, for example, the lipofection method described by Felgner, et
al., Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 .mu.g of
BACULOGOLD.TM. virus DNA and 5 .mu.g of the pBacLP plasmid are
mixed in a sterile well of a microtiter plate containing 50 .mu.l
of serum-free Grace's medium (Invitrogen). Afterwards, 10 .mu.l
Lipofectin plus 90 .mu.l Grace's medium are added, mixed and
incubated for 15 minutes at room temperature. Then the transfection
mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711)
seeded in a 35 mm tissue culture plate with 1 ml Grace's medium
without serum. The plate is rocked back and forth to mix the newly
added solution. The plate is then incubated for 5 hours at
27.degree. C. After 5 hours the transfection solution is removed
from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum is added. The plate is put back into an
incubator and cultivation is continued at 27.degree. C. for four
days.
[0202] After four days the supernatant is collected and a plaque
assay is performed. An agarose gel with "Blue Gal" (Invitrogen) is
used to allow easy identification and isolation of gal-expressing
clones, which produce blue-stained plaques. (A detailed description
of a "plaque assay" of this type can also be found in the user's
guide for insect cell culture and baculovirology distributed by
Invitrogen). After appropriate incubation to allow for plaque
growth, blue stained plaques are picked with a sterile
micropipettor tip. The agar containing the recombinant viruses is
then resuspended in a microcentrifuge tube containing 200 .mu.l of
Grace's medium and the suspension containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are
harvested and stored at 4.degree. C.
[0203] To verify the expression of the LP polypeptide of interest,
Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus expressing the polypeptide of interest at a
multiplicity of infection ("MOI") of about 2. Six hours later the
medium is removed and is replaced with SF900 II medium minus
methionine and cysteine (Invitrogen). If radiolabeled polypeptides
are desired, 42 hours later, 5 mCi of .sup.35S-methionine and 5 mCi
.sup.35S-cysteine (Amersham) are added. The cells are further
incubated for 16 hours and then they are harvested by
centrifugation. The polypeptides in the supernatant as well as the
intracellular polypeptides are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled). Microsequencing of the amino
acid sequence of the amino terminus of purified polypeptide can be
used to determine the amino terminal sequence of the mature
polypeptide and thus the cleavage point and length of the secretory
signal peptide.
Example 4
Cloning and Expression of LP polypeptide in Mammalian Cells
[0204] A typical mammalian expression vector contains at least one
promoter element (which mediates the initiation of transcription of
mRNA), the polypeptide coding sequence, and signals required for
the termination of transcription and polyadenylation of the
transcript. Additional optional elements include enhancer(s), a
Kozak sequence and an intervening sequence (intron) flanked by
donor and acceptor sites for RNA splicing. Highly efficient
transcription initiation can be achieved with the early and late
promoters from SV40, the long terminal repeats (LTRs) from
retroviruses (e.g., RSV, HTLV I, HIV) and the early promoter of the
cytomegalovirus (CMV). However, cellular promoters can also be used
(e.g., the human actin promoter). Suitable expression vectors for
use in practicing the present invention include, but are not
limited to, pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or PLNCX
(Clontech), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-)
(Invitrogen), PSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat
(ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC67109).
Suitable mammalian host cells include, but are not limited to,
human Hela 293 (ATCC CRL-1573), H9 (ATCC HTB-176), Jurkat cells
(ATCC CRL-1990), mouse NIH3T3 (ATCC HB11601), C127 cells (ATCC
CRL-1804), Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells
(ATCC CCL-1) and Chinese hamster ovary (CHO) cells (ATCC
CCL-61).
[0205] Alternatively, the nucleic acid encoding the polypeptide of
interest is expressed in stable cell lines that contain the nucleic
acid integrated into a host chromosome. The co-transfection of the
nucleic acid encoding the polypeptide of interest along with a gene
encoding a selectable marker such as DHRF (dihydrofolate
reductase), GPT neomycin, or hygromycin allows the identification
and isolation of the transfected cells.
[0206] The transfected gene can also be amplified to express large
amounts of the encoded polypeptide. The DHFR marker is useful to
develop cell lines that carry several hundred or even several
thousand copies of the gene of interest. Another useful selection
marker is the enzyme glutamine synthase (GS) (Murphy, et al.,
Biochem. J. 227: 277-279 (1991); Bebbington, et al., BioTechnology
10: 169-175 (1992)). Using these markers, the mammalian cells are
grown in selective medium and the cells with the highest resistance
are selected. These cell lines contain the amplified gene(s)
integrated into a chromosome. Chinese hamster ovary (CHO) and NSO
cells are often used for the production of polypeptides.
[0207] The expression vectors pC1 and pC4 contain the strong LTR
promoter of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell.
Biol. 5: 438-447 (1985)) plus a fragment of the CMV-enhancer
(Boshart, et al., Cell 41: 521-530 (1985)). Multiple cloning sites
(e.g., with the restriction enzyme cleavage sites BamH I, Xba I and
Asp 718), facilitate the cloning of the gene of interest. The
vectors contain, in addition to the 3' intron, the polyadenylation
and termination signal of the rat preproinsulin gene.
Example 5
Cloning and Expression of LP Polypeptides in COS Cells
[0208] The expression vector containing the nucleic acid encoding
the LP polypeptide of interest, is made by cloning a cDNA encoding
the LP polypeptide of interest into the expression vector
pcDNAI/Amp or pcDNAIII (Invitrogen).
[0209] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of prokaryotic cells containing plasmid; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a multiple cloning site polylinker, an SV40 intron;
(5) several codons encoding a hemagglutinin fragment (i.e., an "HA"
tag to facilitate purification) or HIS tag (see, e.g, Ausubel,
supra) followed by a termination codon and polyadenylation signal
arranged so that a cDNA can be conveniently placed under expression
control of the CMV promoter and operably linked to the SV40 intron
and the polyadenylation signal by means of restriction sites in the
polylinker. The HA tag corresponds to an epitope derived from the
influenza hemagglutinin polypeptide described by Wilson, et al.,
Cell 37: 767-778 (1984). The fusion of the HA tag to the target
polypeptide allows easy detection and recovery of the recombinant
polypeptide with an antibody that recognizes the HA epitope.
pcDNAIII contains, in addition, the selectable neomycin marker.
[0210] A DNA fragment encoding the LP polypeptide of interest is
cloned into the polylinker region of the vector so that recombinant
polypeptide expression is directed by the CMV promoter to which it
is operably linked. The plasmid construction strategy is as
follows. The LP cDNA of a clone is amplified using primers that
contain convenient restriction sites.
[0211] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with suitable restriction enzyme(s) and then ligated.
The ligation mixture is transformed into E. coli (e.g., strain
SURE, Stratagene, La Jolla, Calif.), and the transformed culture is
plated on ampicillin LB media plates which then are incubated to
allow growth of ampicillin resistant colonies. Plasmid DNA is
isolated from resistant colonies and examined by restriction
analysis or other means for the presence of the LP
polypeptide-encoding fragment.
[0212] For expression of recombinant LP121A, LP121B, LP121C or
LP10034, COS cells are transfected with an expression vector, as
described above, using e.g., DEAE-DEXTRAN. Cells are incubated
under conditions appropriate for expression of LP121A, LP121B,
LP121C or LP10034 by the vector.
[0213] Expression of the LP121A-HA, LP121B-HA, LP121C-HA or
LP10034-HA fusion polypeptide is detected by radiolabeling and
immunoprecipitation, using methods described in, for example
Harlow, et al., Antibodies: A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To
this end, two days after transfection, the cells are labeled by
incubation in medium containing .sup.35S-cysteine for 8 hours. The
cells and the media are collected, and the cells are washed and
lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40,
0.1% SDS (sodium dodecyl sulfate), 0.5% DOC (deoxycholate), 50 mM
TRIS, pH 7.5, as described by Wilson, et al. cited above. Proteins
are purified from the cell lysate and from the culture media using
a HA-specific monoclonal antibody. The purified polypeptides then
are analyzed by SDS-PAGE and autoradiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
Example 6
Cloning and Expression of LP Polypeptide in CHO Cells
[0214] The vector pC4 is used for the expression of LP polypeptide
of interest in CHO cells. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains
the mouse DHFR gene under control of the SV40 early promoter. CHO
cells or other cells lacking dihydrofolate activity that are
transfected with these plasmids can be selected by growing the
cells in a selective medium (alpha minus MEM, Invitrogen)
supplemented with methotrexate. The amplification of the DHFR genes
in cells resistant to methotrexate (MTX) has been well documented
(see, e.g., F. W. Alt, et al., J. Biol. Chem. 253: 1357-1370
(1978); J. L. Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:
107-143 (1990); and M. J. Page and M. A. Sydenham, Biotechnology 9:
64-68 (1991)). Cells grown in increasing concentrations of MTX
develop resistance to the drug by overproducing the target enzyme,
DHFR, as a result of amplification of the DHFR gene. If a second
gene is linked to the DHFR gene, it is usually co-amplified and
over-expressed. It is known in the art that this approach can be
used to develop cell lines carrying more than 1,000 copies of the
amplified gene(s). Subsequently, when the methotrexate is
withdrawn, cell lines are obtained which contain the amplified gene
integrated into one or more chromosome(s) of the host cell.
[0215] Plasmid pC4 contains the LTR strong promoter of the Rous
Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5: 438-447
(1985)) plus a fragment isolated from the enhancer of the immediate
early gene of human CMV (Boshart, et al., Cell 41: 521-530 (1985).
Downstream of the promoter are BamH I, Xba I, and Asp 718
restriction enzyme cleavage sites that allow insertion of the
genes. Downstream of these cloning sites the plasmid contains the
3' intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for expression,
(e.g., human b-actin promoter, SV40 early or late promoters, or the
LTR from other retroviruses). Clontech's Tet-Off and Tet-On gene
expression systems and similar systems can be used to express the
LP polypeptide of interest in a regulated way in mammalian cells
(M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551
(1992)). For the polyadenylation of the mRNA, polyadenylation
signals, (e.g., from the human growth hormone or globin genes) can
be used. Stable cell lines carrying a gene of interest integrated
into the chromosomes can also be selected upon co-transfection with
a gene expressing a selectable marker such as gpt, G418 or
hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
[0216] The plasmid pC4 is digested with appropriate restriction
enzyme(s) and then dephosphorylated using calf intestinal
phosphatase by procedures known in the art. The vector is then
isolated from a 1% agarose gel.
[0217] The DNA sequence encoding the LP polypeptide of interst is
amplified using PCR oligonucleotide primers corresponding to
sequences 5' and 3' to the sequence of interest.
[0218] The amplified fragment is digested with suitable
endonuclease(s) and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli (e.g., HB101 or XL-1 Blue cells) are
then transformed and bacteria are identified that contain the
fragment inserted into plasmid pC4 using, for instance, using
restriction enzyme analysis.
[0219] Chinese hamster ovary (CHO) cells lacking an active DHFR
gene are used for transfection. Five micrograms of the expression
plasmid pC4 is cotransfected with 0.5 .mu.g of the plasmid pSV2-neo
using e.g., lipofectin. The plasmid pSV2-neo contains a dominant
selectable marker, the neomycin resistance gene from Tn5 encoding
an enzyme that confers resistance to a group of antibiotics
including G418. The cells are seeded in alpha minus MEM
supplemented with 1 .mu.g/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of methotrexate plus 1 .mu.g/ml G418. After about 10-14 days clonal
colonies are independently trypsinized and then seeded in 6-well
petri dishes or 10 ml flasks using different concentrations of
methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
independently transferred to a new well of a 6-well plate
containing even higher concentrations of methotrexate (1 mM, 2 mM,
5 mM, 10 mM, 20 mM). The same procedure is repeated until clones
are obtained which grow at a concentration of 100-200 mM
methotrexate. Expression of the desired gene product is analyzed,
for instance, by SDS-PAGE and Western blot or by reverse phase HPLC
analysis.
Example 7
Overexpression of huJAM1, huJAM2, huJAM3, LP121A, LP121B, LP121C
and LP10034 in Xenopus Embryos
[0220] Injecting RNA that encode novel proteins into early Xenopus
laevis embryos may result in various phenotypes in the early
tadpoles (i.e. split axis, anteriorization etc.) that help
elucidate the function of these proteins.
[0221] The DNA encoding the LP polypeptide of interest is first
cloned into an expression vector (e.g., PR1) containing the T7 RNA
polymerase promoter. Plasmid DNA containing the LP cDNA insert is
linearized with Not I restriction enzyme (Gibco BRL, #15441-025)
and in vitro transcribed using MESSAGE MACHINE (Ambion, #1344)
containing T7 RNA polymerase. The RNAs produced are examined on a
1.25% agarose gel to confirm success of the transcription reaction
as well as the appropriate size and concentration of the RNA
product. The RNA is translated in vitro using the biotin in vitro
translation kit (Roche, #1559451) to determine if RNA is of
sufficient quality to produce protein of predicted molecular
weight. Finally, RNA is diluted to 200 .mu.g/ml with Rnase-free
water and stored at -20.degree. C. until ready for microinjection
into Xenopus embryos.
[0222] Female Xenopus frogs are injected with 300 U/frog of human
choronic gonadotropin (Sigma Corp., St. Louis, Mo.) to induce egg
laying. Eggs are harvested from the females and combined with
macerated testis to fertilize the eggs in vitro.
[0223] Fertilized eggs are dejellied with 2% cysteine in water (pH
8.0), rinsed, and transferred to 1.times.MR (0.1 M NaCl, 1.8 mM
KCl, 2.0 mM CaCl.sub.2, 1.0 mM MgCl.sub.2 and 50 mM HEPES-NaOH).
Embryos develop at room temperature or 15.degree. C. until signs of
the first cleavage furrow appear. Embryos are then transferred to
4% Ficoll (Sigma Corp.) in 1.times.MR prior to injection.
[0224] Five nl (1 ng) of RNA is injected into both cells of a
two-cell stage embryo. Uninjected controls are also maintained.
Embryos are left in 4% Ficoll/1.times.MR solution until they reach
the blastula stage of development, then they are switched to
0.1.times.MR with 4% Ficoll and continue to develop at 18.degree.
C. Embryos are observed for morphological effects during the next
2-3 days. Phenotypes of tadpoles are recorded by photography using
a digital camera and a dissecting microscope.
[0225] Histological analysis of embryos is also performed. The
tadpoles are fixed in 3% formaldehyde and embedded in paraffin.
Tissues are prepared for analysis by removing the parafin with
xylene and then gradually rehydrating the tissue with graded
solutions of ethanol and water. Sections are then stained with
hematoxylin and eosin solutions, coverslipped and viewed under a
light microscope. Compared to control embryos of three day old
tadpoles, the embryos injected with full-length huJAM2 have
enlarged dilated hearts.
[0226] Animal Cap Assays with LP Polypeptides
[0227] Animal cap explants from early Xenopus embryos normally give
rise to cells solely of the ectodermal lineage. Addition or
expression of foreign proteins can change the fate of these cells
into other lineages (endodermal and mesodermal) thereby helping to
functionate newly discovered proteins.
[0228] RNA-injected embryos at stage 8 (Niewkoop and Faber, 1967)
are harvested for animal cap studies. Vitelline membranes are
removed from the embryos and a sheet of cells from the animal pole
(animal cap) is isolated. The animal caps are grown in a 96-well
tissue culture dish (Costar) with animal cap buffer [0.2.times.MR,
1% BSA (Sigma Corp.), and 59 .mu.g/ml Gentamicin (Sigma Corp.)]
overnight at room temperature until control embryos reach
approximately stage 17-19 (Niewkoop and Faber, 1967).
[0229] In addition, animal cap assays can also be performed with
uninjected embryos with addition of the LP protein (1-20 nM) to the
animal cap buffer.
[0230] Results of these assays are monitored by morphology, RT-PCR
for tissue specific gene expression, and immunohistochemistry for
visualization of tissue type and distribution within the
explant.
[0231] Overexpression of full-length, membrane-bound HuJAM2 (SEQ ID
NO: 2) results in embryos with ventral edema and dilated hearts.
Overexpression of the truncated, extracellular form of huJAM2
[LP121A (SEQ ID NOs: 11)] or the truncated, extracellular form of
huJAM3 [LP10034 (SEQ ID NO:14)} results in no observable altered
phenotype in this assay. Since only overexpression of the
full-length, membrane-bound HuJAM results in heart edema phenotype
in the Xenopus embryo, it is contemplated that this molecule is
involved in diseases of the heart in humans. HuJam display
homotypic and/or heterotypic binding; e.g., it is known that huJAM2
has both types of binding. Extracellular huJAM, e.g., LP121A,
LP121B, LP121C, LP10034, are able to neutralize or block binding of
protein molecules to the overexpressed full-length, membrane-bound
huJAM in diseased states of the heart. This blocking or
neutralization is accomplished by extracellular huJAM binding the
extracellular portion of a membrane-bound huJAM and preventing it
from interacting with receptors and/or ligands that exist on other
cells or that are soluble in the extracellular space. This blocking
or neutralization prevents, reverses, and/or treats the diseased
heart condition.
Example 8
Tissue Distribution of LP mRNA
[0232] Non-radioactive northern blot analysis is performed to
examine gene expression in human tissues. First, a control probe,
Human G3PDH cDNA Control Probe (Clontech #9805-1), is labeled with
digoxigenin using DIG-High Prime Labeling kit (Roche #1585606).
[0233] The cDNA encoding the LP polypeptide of interest is cloned
into a pPR1 or XenoFLIS vector using PCR conditions known to a
person skilled in the art. This may be accomplished using the
following PCR mixture: 5 .mu.L PCR buffer and 5 .mu.L PCR DIG mix
from PCR DIG Probe Synthesis Kit (Roche # 1636090); 0.75 .mu.L
Enzyme Mix (Expand High Fidelity, Roche #1732641); 5 .mu.L of 10
.mu.M pPR1 probe primer (5'-TGCAAAGCTTGGCGCGCC-3- ' SEQ ID NO: 27);
5 .mu.L of 10 .mu.M FLAG Probe Primer
(5'-CTTGTCGTCGTCATCCTTGTAGTCG-3' SEQ ID NO: 28); 1 .mu.L plasmid
DNA (long); and 28.25 .mu.L sterile water. The cDNA is labeled
using this mixture and the following PCR method: one cycle of
95.degree. C. for two minutes; thirty to forty cycles of 95.degree.
C. for thirty seconds, 60.degree. C. for thirty seconds, and
70.degree. C. for 1.5 minutes/1 kb of inserted cDNA; one cycle of
72.degree. C. for ten minutes; and a 4.degree. C. soak cycle.
[0234] Next, the samples are hybridized to human multiple tissue
northern (MTN) blot membranes (Clontech #7780-1) human
cardiovascular system MTN blot (Clontech #7791-1) and human
endothelial cells, diseased, and normal heart using DIG Easy Hyb
(Roche # 1603558). Membranes are prehybridized with DIG Easy Hyb
for 1 hour at 50.degree. C. The DIG DNA probes prepared above are
denatured at 100.degree. C. for 10 minutes then immediately placed
on ice, diluted with DIG Easy Hyb (2 .mu.l probe to 10 ml Easy
Hyb), and the membranes are incubated overnight at 50.degree. C.
with the diluted hybridization/probe mixture.
[0235] Detection of the membranes is accomplished using CDP-Star
chemiluminescent substrate (Roche # 1685627) and X-ray film. The
membranes are washed twice in 2.times.SSC at 5.degree. C. for 30
minutes each, twice in 0.5.times.SSC at 50.degree. C. for 30
minutes each and twice in 0.1.times.SSC at 50.degree. C. for 30
minutes each. The membranes are blocked with DIG Blocking Buffer
(Wash and Block Buffer Kit, Roche # 1585762) for one hour to
overnight at room temperature, and anti-digoxigenin-AP Fab
fragments (Roche # 1093274) are then added at a 1:20,000 dilution
for 2 hours to overnight at room temperature. The membranes are
washed three times in 1.times. wash buffer, equilibrated in
detection solution (DIG Wash and Block Buffer Kit, Roche # 1585762)
then incubated with CDP-Star (Roche # 1685627) at 1:100 dilution
for 5 minutes at room temperature. After lightly blotting, the
membranes are placed between two pieces of transparency film and
exposed to X-ray film for various exposure times to ensure
detection of all possible bands.
[0236] Northern blot data indicate that the main tissue of LP121A
expression is the heart and placenta, with lesser amounts present
in brain, smooth muscle, kidney, small intesting, and lung tissues
and even less still in the colon, thymus, spleen and liver. All
areas of the heart express huJAM2. Fetal heart appears to have a
more intense signal compared to adult heart. Thus, the DNA encoding
extracellular huJAM and their respective mRNA and proteins are
contemplated to be useful in the treatment of cardiovascular
diseases. In addition, huJAM2 may be involved in inflammatory
diseases of the kidney, small intestine, lung, colon and liver.
[0237] Human Endothelial, Diseased, and Normal Heart Northern
Blots
[0238] Total RNA is isolated using the RNAqueous-Midi Kit (Ambion,
#1911) following the manufacturer's protocol. Twenty micrograms of
RNA, diluted in 3 volumes of NorthernMax formaldehyde loading dye
(Ambion, #8552) is separated with a precast, 1.25% agarose,
1.times.MOPS RNA gel (BioWhittaker, #52922). Gel is stained for RNA
integrity with 0.5 ug/ml of ethidium bromide for 20 min. and
destained with distilled water for 10 min. RNA is transferred to a
BrightStar-Plus membrane (Ambion, #10102) using NorhternMax
Transfer Buffer (Ambion, #8672). RNA is crosslinked and stored at
-20.degree. C. until ready to hybridize by methods already
described above.
[0239] Homotypic Binding Assays
[0240] A BIAcore 2000 instrument is used to detect real-time
binding between immobilized LP121A, LP121B, LP121C or LP10034 and
soluble LP121A, LP121B, LP121C or LP10034. LP121A, LP121B, LP121C
or LP10034 is diluted to a concentration of 50 .mu.g/mL in 10 mM
sodium acetate buffer at pH 5.0. LP121A, LP121B, LP121C or LP10034
is immobilized to a CM5 sensor chip using the amine coupling
method.
[0241] LP121A, LP121B, LP121C and LP10034 proteins are diluted in
HBS-EP buffer. Samples are injected over LP121A, LP121B, LP121C or
LP10034 and control surfaces using the kinject method. Samples
containing 5 .mu.g/mL and 1 .mu.g/mL of protein are injected at 30
.mu.L/min for three minutes with a ninety second dissociation time.
LP121A, LP121B, LP121C and LP10034 and control surfaces are then
regenerated with glycine-hydrochloride at pH 3.0.
Example 9
Protein Binding in Human Tissue
[0242] Binding of LP121A, LP121B, LP121C and LP10034 proteins to
human tissues is determined by protein staining with fluorescent
dye. The following human tissues are used in this assay:
3 TABLE 3 LP polypeptide Tissue LP121A LP10034 Kidney 0 0 Liver 0 0
Heart 0 0 Lung 0 0 Spleen 0 0 Pancreas 1 1 Gut 1 1 thymus 0 0
Ampulla 0 0 Bone marrow nd 2 Prostate 1 0 Ovary 1 0 Skin 0 0
Vessels 0 0 Skeletal Muscle 0 0 Brain 0 0 Peri nerve 0 0 Breast 0 0
Lymph node 2 0 Adipose 0 0 Adr. Gland 0 0
[0243] All tissues are fixed with 3% paraformaldehyde and embedded
in paraffin. Tissues are prepared for analysis by removing the
paraffin with xylene then gradually rehydrating the tissue with
graded solutions of ethanol and water. Antigen retrieval is
performed to unmask antigenic sites so that antibodies can
recognize the antigen. This is accomplished by soaking the tissue
in citrate buffer (Dako, Carpinteria, Calif.) for twenty minutes at
80 to 90 degrees C. followed ten minutes at ambient temperature.
The tissue is then washed in tris-buffered saline (TBS) containing
0.05% TWEEN.RTM.-20 and 0.01% thimerosol. To minimize non-specific
background staining, the tissue is soaked in non-serum protein
block (Dako) for forty-five minutes, after which the protein block
is removed by blowing air over the tissue.
[0244] The tissue is exposed for two hours to the FLAG-HIS tagged
LP121A, LP121B, LP121C or LP10034 protein at 10 .mu.g/mL. Following
exposure, the tissue is washed twice with tris-buffered saline
(TBS) containing 0.05% TWEEN.RTM.-20 and 0.01% thimerosol. The
tissue sample is then incubated for one hour with mouse anti-FLAG
antibody at 10 .mu.g/mL. Subsequently, the tissue is washed twice
with tris-buffered saline (TBS) containing 0.05% TWEEN.RTM.-20 and
0.01% thimerosol. Next, the tissue is exposed to rabbit anti-mouse
Ig with Alexa 568, a fluorescent dye, at 10 .mu.g/mL for one hour,
followed again by two washes with TBS containing 0.05%
TWEEN.RTM.-20 and 0.01% thimerosol. Finally, the tissue is
coverslipped with fluorescence mounting media, and the fluorescence
is measured. A positive fluorescence reading indicates that the
protein binds with antigens on the tissue, suggesting that the
protein binds to that tissue indicating localization of possible
ligand or receptor for that LP polypeptide.
Example 9
In Vivo Function of LP121A, LP121B, LP121C or LP10034 with
Endotoxin Challenged Mice
[0245] Endotoxin is a lipopolysaccharide (LPS) from gram negative
bacterial cells which immediately induces systemic release of
inflammatory mediators such as TNF and IL-1. Mice injected with LPS
usually die within 48 hours of injection. DNA injected into the
tail veins of mice will translocate to the liver where protein
encoded by the DNA will be produced and secreted into the blood
stream. Injection of Il-10 DNA protects mice from death when
challenged with LPS. Injection of LP121A, LP121B, LP121C or LP10034
will test their anti-inflammatory properties.
[0246] Plasmid DNA for LP121A, LP121B, LP121C or LP10034 under
control of the CMV promoter is prepared from DH5-.alpha. E. coli
cells using an endotoxin free DNA isolation kit (Qiagen #12362).
Il-10 is cloned behind a CMV promoter in the pcDNA3.1/v5-His-TOPO
vector (Invitrogen #K4800-01) as a positive control DNA (Il-10
abrogates the effects of the endotoxin). DNA is quantified using a
spectrophotometer and prepared for injection with the TRANSIT in
vivo gene delivery kit (Mirus Corp). Mice (Harlan) aged 6-8 week
and weighing on average 24.5 g are injected with 20 ug/mouse (in
2.4 mls) of LP121A, LP121B, LP121C, LP10034, IL-10 or vector alone
DNA into the tail vein. Twenty-four hours post-DNA injection mice
are challenged with endotoxin and D-galactosamine (10 .mu.g/mouse
and 6 mg/mouse respectively). Mice are monitored 3.times. daily for
72 hours to determine survival (FIG. 17). Two hours post LPS
injection, retro-orbital bleeds are done to collect serum for
analysis of cytokines by Luminex (BioRad, #171-F12080)(FIG.
18).
[0247] It is contemplated that the LP121A DNA injected into the
mice is transcribed then translated into protein and secreted into
the blood stream. It is further contemplated that the mice injected
with LP121A survive the LPS effect by reducing or eliminating the
inflammatory response resulting from the LPS. Since vector alone
does not protect the mice from the LPS challenge, it is concluded
that the LP polypeptide protects the mice from death in this assay.
It is further contemplated from this data that full-length,
transmembrane huJAM2 is involved in the transmigration of immune
cells from the blood stream into the tissues and that extracellular
huJAM inhibit this function of full-length huJAM.
[0248] In addition, LP121A protects mice from death without
altering cytokine secretion in the animals. Alterations in cytokine
expression levels can abate inflammatory responses. Based on the
hypothesis for the role of LP polypeptides of the invention in
inhibiting inflammation, no changes in assayed cytokine levels is
consistent with this hypothesis. Mice injected with LP121A
(extracellular huJAM2) DNA (SEQ ID NO. 16) are protected from death
(5/5 mice survived) when challenged with LPS. The graph in FIG. 17
shows one experiment of two performed in the LPS model. In the
second experiment performed, LP121A proteced 80 of the mice (4/5
mice survived), vector alone protected 0% of the mice, and IL-10
protected 100% of the mice 48 hours after LPS injection. LP121A
protected the mice from death without increasing cytokine levels
(such as TNF-.alpha.) in the blood stream.
Example 10
In Situ Assays
[0249] Human Peripheral Blood T Cell Assay
[0250] Tissue culture plates (Nunc) are coated with 0.5 .mu.g/ml or
50 ng/ml anti-human CD3 monoclonal antibody (Pharmingen, #30111A)
overnight at 4.degree. C. Next, varying concentrations (1-3
.mu.g/ml) of Lp121A, LP121B, LP121C, LP10034, or CD28 monoclonal
antibody (Pharmingen, #33741A) are added for 4 hours. Human blood
was diluted 2-fold with RPMI-1640(Gibco BRL, #22400) and is
separated over a Ficoll (Sigma, #F8636) gradient by centrifugation
at 2000 rpm for 5 min. Cells are collected from the gradient and
washed twice in RPMI-1640 and centrifuge at 1000 rpm for 5 min.
Cells are resuspeded in 0.5% BSA/PBS. T cells are isolated using
the Miltenyi BioTec Pan T cell Isolation kit (530-01). Cells are
washed in media, suspended in RPMI-1640 with 10% FCS. Cells are
plated at 2.times.10.sup.6 cells/ml in 100 ul/well, incubated for 3
days at 37.degree. C. with 5% CO.sub.2. Supernatants are collected
to measure cytokine analysis (Il-beta1, Il-2, Il-4, Il-5, Il-10,
GMCSF, IFN-gamma, and TNF-alpha) with Luminex kits
(BioRad,#171-A12080) following the manufacturer's instructions.
Then 100 .mu.l of medium is added back to the cells and cells are
pulsed with 1 .mu.Ci of .sup.3H-thymidine overnight at 37.degree.
C. with 5% CO.sub.2. Next, cells are frozen and thawed for lysis
and harvested with a Skatron Semiautomatic cell harvester. Total
.sup.3H-thymidine incorporation is measured by a beta-scintillation
counter, with mean of triplicate results reported.
[0251] Mouse T-cell Assay
[0252] Tissue culture plates are coated with 0.5 .mu.g/ml of
anti-mouse CD3 monoclonal antibody (Pharmingen, #30111A) in DPBS by
adding 100 .mu.l/well overnight at 4.degree. C. Wells are then
washed with DPBS and coated with 0.23 .mu.g/ml of anti-CD28
monoclonal antibody. Various concentrations (1-3 .mu.g/ml) of
LP121A, LP121B, LP121C or LP10034 are added to the plates for 4
hours at 37.degree. C. Mouse T cells are harvested from lymph nodes
and spleens from 4 Balb/c mice. Tissue is ground between 2 frosted
glass slides and pipetted through a 70 .mu.M nylon cell strainer
into a 50 ml tube. Cells are washed 3.times. with RPMI-1640 with
10% FCS and centrifuged at 1000 rpm for 5 min. Cells are
resuspended in DPBS with 0.5% BSA. Murine T cells are positively
selected using CD90 microbeads and autoMACS system (Miltenyi).
Cells are resuspended to 2.times.10.sup.6 cells/ml in RPMI-1640
with 10% FCS and antibiotic. One hundred microliters of cell
suspension is added to each well of a 96-well plate. Cells are
incubated for 3 days, then supernatants are collected to measure
cytokine release. One hundred microliters of medium is added back
to the cells and the cells are pulsed with 1 .mu.Ci
.sup.3H-thymidine for 8 hours. Next, the cells are frozen and
thawed for lysis and harvested with a Skatron Semiautomatic cell
harvester. Total .sup.3H-thymidine incorporation is measured by a
beta-scintillation counter, with mean of triplicate results
reported.
[0253] RAW264.7 Bioassay RAW264.7 cells are plated at
1.times.10.sup.5 cells/well in a 96 well plate in 200 .mu.l final
volume in DMEM (GibcoBRL, #11995) with 10% fetal bovine serum
(FBS). Cells are incubated overnight at 37.degree. C. and 5%
CO.sub.2, then LP121A, LP121B, LP121C or LP10034 are added at 1
.mu.g/ml for 6 hours at 37.degree. C. and 5% CO.sub.2. Next, the
cells are incubated with or without LPS (Sigma, 100 ng/ml) for an
additional 24 hours. Cytokines are measured 24 hours after LPS
addition using 30 .mu.l of supernatant and the BioPlex Mouse
Cytokine Assay (BioRad, #171-F12080). Nitric oxide is measured at
40 hours after LPS addition using 50 .mu.l of supernatant and
Griess Reagent (Sigma Corp., #G4410).
[0254] A549 Bioassay
[0255] S549 cells are plated at 3.times.10.sup.4 cells/well in a 96
well plate in 200 .mu.l final volume in HamsF12K (GibcoBRL,
#11765-054) with 10% FBS and 0.15% sodium bicarbonate. Cells are
incubated 24 hours at 37.degree. C. and 5% CO.sub.2 then starved
for 16-18 hours in serum free medium. LP121A, LP121B, LP121C or
LP10034 are added to the medium and incubated 6 hours at 37.degree.
C. before adding TNF-alpha (R&D Systems, #210-TA-010, 50
ng/ml). Cells are incubated for an additional 24 hours at
37.degree. C., then 10 .mu.l of supernatant is used for cytokine
detection using the BioPlex Human Cytokine Assay (BioRad,
#171-A12080). Il-8 ELISAs (R&D System, #D8050) use 5 .mu.l of
supernatant.
[0256] HUVEC Bioassay
[0257] HUVEC cells are plated at 4.times.10.sup.4 cells/well in a
96 well plate in 200 .mu.l EGM (Clonetics). Cells are incubated for
24 hours at 37.degree. C. then starved for 16-18 hours in serum
free medium (EGM with no supplements). LP121A, LP121B, LP121C or
LP10034 are added (1 .mu.g/ml) and incubated for 6 hours at
37.degree. C. before adding TNF-alpha (R&D Systems,
#210-TA-010, 10 ng/ml). Cells are incubated an additional 24 hours,
then cytokines are measured using 15 .mu.l of supernatant and
BioPlex Human Cytokine Assay (BioRad,# 171-A12080).
[0258] Primary Macrophage Bioassay
[0259] Mice are injected with 3 ml of 3% Brewer thioglycollate
intraperinoneally 72 hours before harvesting peritoneal
macrophages. Mice are sacrificed and 10 ml of DMEM+10% FBS is
injected into the peritoneum. The solution is aspirated after
gentle washing. The cells are plated at 3.times.10.sup.5
cells/well, 200 .mu.l volume in DMEM+10% FBS in a 96 well plate.
Cells are incubated for 2 hours at 37.degree. C. then medium is
removed and LP121A, LP121B, LP121C or LP10034 is added (diluted in
DMEM+10% FBS). Cells are incubated an additional 24 hours at
37.degree. C. then cytokines are measured using 30 .mu.l of
supernatant and the Mouse BioPlex Cytokine Assay (BioRad,
#171-F12080).
[0260] Adhesion Assays
[0261] One hundred microliters of anti-FLAG antibody is coated onto
a 96 well plate at 5 .mu.g/ml in PBS. Next, 5 .mu.mol of LP121A,
LP121B, LP121C, LP10034 (each fused to a FLAG epitope), or
irrelevant FLAG-tagged protein is captured by the bound anti-FLAG
antibody. Various cell lines and human leukocytes are labeled with
calcein (Molecular Probes, Eugene, Oreg.) at 50 .mu.g/ml for 25
min. at 37.degree. C. Cell binding is performed for 90 min. at
37.degree. C. with 2.5.times.10.sup.5 cells/well in binding buffer
(Tris-buffered saline, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, and 1 mM
MnCl.sub.2). Wells are washed three times with binding buffer and
lysed with 50 mM Tris (pH 7.5), 5 mM EDTA, and 1% IGEPAL.
Fluorescence is read in a Cytofluor 4000 (Perseptive Biosystems)
with excitation at 485/20 nm and emission at 530/25 nm. Specific
binding is determined by removal of values obtained by the
irrelevant FLAG tagged protein.
Example 11
Transendothelial Cell Migration Assay
[0262] Isolation of Monocytes and Neutrophils
[0263] Whole human blood is collected into a heparinized syringe by
a phlebotomist. The blood is then carefully layered over
Polymorphprep solution (Nycomed Pharma As) at a ratio of 40 ml
blood to 10 ml Polymorphprep in a 50 ml conical centrifuge tube.
The tube is then centrifuged at 500.times.g for 30 minutes in a
swinging bucket rotor at 20.degree. C. At the conclusion of the
run, two bands of leukocytes are present. The top band contains the
mononuclear cells from which the monocytes will be isolated. The
lower band contains primarily neutrophils. The top band is
carefully aspirated and diluted 10.times. with RPMI-1640 (Gibco
BRL, #22400). The lower band is then aspirated and diluted
10.times. with RPMI-1640. Both populations are washed twice with
RPMI-1640 and placed on ice. The neutrophils are now ready for
counting and loading.
[0264] Monocytes are further isolated using the AutoMACS sorting
system and monocyte isolation kit (Miltenyi Biotec). According to
the protocol, 10,000,000 cells are suspended in 60 .mu.l PBS/BSA
(0.5%)/EDTA (2 mM). The suspension then receives 20 ul Fc blocking
reagent and 20 .mu.l antibody-hapten cocktail. This cocktail labels
all cells except monocytes. After a 20 min. 6.degree. C.
incubation, the cells are washed one time and resuspended in 60 ul
buffer. The suspension then receives 20 ul Fc blocking reagent and
20 .mu.l anti-hapten microbeads. After a 20 min., 6.degree. C.
incubation, the cells are washed one time and resuspended in 1.0 ml
buffer. The cells are then run through the AutoMACS cell sorter and
the negative fraction used as the monocyte population.
[0265] Loading of both the neutrophils and monocytes is
accomplished using calcein AM from Molecular Probes. Cells are
suspended in RPMI-1640. The suspension receives pluronic F-127 and
calcein AM at final concentrations of 0.1% and 5 uM. The cells are
incubated at room temperature for 30 minutes. After washing twice,
the fluorescent cells can be resuspended in the buffer of choice
for assay.
[0266] Chemotaxis Through Cell Monolayers
[0267] Human umbilical vein endothelial cells (HUVECs) are cultured
in Transwells (8 .mu.m pore, Costar) and grown to confluence.
Soluble LP121A, LP121B, LP121C or LP10034 are added at various
concentrations (0.2-5 .mu.g/ml) to HUVEC monolayer, neutrophils, or
monocytes for 30 min. at 37.degree. C. Labeled monocytes and
neutrophils are diluted to a concentration of 1.8.times.10.sup.6
cells/ml and 750 .mu.l of cells are added/well for 60 min. at
37.degree. C. Chemokines MCP-1 and/or MCP-3 (R&D Systems,
#279-MC-010 and #282-P3-010) may be added to the lower chamber at
100 ng/ml. The number of labeled cells in the bottom chamber is
measured using the Cytofluor 4000 (Perseptive Biosystems).
[0268] Liquid Bone Marrow Assay
[0269] Human bone marrow cells (Poieteics) are cultured in IMDM+30%
FBS in V-bottom polyproylene 96 well plates for 10-11 days in the
presence of differing combinations of stem cell factor (R&D
systems #255-SC-010, 10 ng/ml), Il-3 (R&D Systems #203-IL-010,
0.1 ng/ml), erythropoietin (R&D Systems #286-EP-250, 1 U/ml),
transforming growth factor beta (R&D Systems #240-B-002, 10
ng/ml), macrophage colony stimulating factor (R&D Systems
#216-MC-005, 40 ng/ml), and LP121 or LP10034 (400 ng/ml). Feeding
occurs on days 4 and 8. Cells are centrifuged in plate and
resuspended in 10 .mu.g/ml unconjugated IgG. Suspensions are
incubated at 4.degree. C., with both anti CD14-FITC (1:100,
Miltenyi Biotech) and anti CD36-PE (1:20, Pharmingen, #30985X) for
20 min. Cells are washed with PBS then transferred to flow cuvettes
containing 1 ml PBSA (fv) containing 100 .mu.l Flow Count beads
(Coulter, #7547053). Cell analysis is done by flow cytometry.
Example 12
Soluble HuJAM2 and HuJAM3 Interactions
[0270] Self-association and binding of 2oluble HuJAM3 to 2oluble
HuJAM2 were investigated using analytical ultracentrifuge (AUC),
size-exclusion chromatography (SE-HPLC), and isothermal titration
experiment (ITC), and surface plasma resonance.
[0271] Sedimentation equilibrium experiments were performed using
an XLA analytical ultracentrifuge. A 110 .mu.l aliquot of sample
solution at protein concentration of about 0.2 to 0.4 mg/mL in PBS
buffer was added to the sample column of the cell and 120 .mu.l of
the PBS buffer was added to the reference column of the cell. The
equilibrium run was performed at 20.degree. C. and 16000 rpm
overnight until the system reached equilibrium. At equilibrium, the
system exhibits a concentration gradient along the column. For an
ideal single species, a plot of 1 nC versus r.sup.2 gives a
straight line and the slope is directly related to the molecular
weight of macromolecule by the following equation
M(1-.nu..rho.).omega..sup.2/2RT=dlnC/dr.sup.2=slope
[0272] Where: M is the molecular weight
[0273] .nu. is the partial specific volume (mL/g)
[0274] .rho. is the density of the solvent (g/mL)
[0275] .omega. is rotor speed (radians/second)
[0276] R is gas constant (8.314*10.sup.7 erg/mol K)
[0277] T is temperature in Kelvin
[0278] C is concentration of the solute at radius r
[0279] The density of 1.00 g/cm.sup.3 for PBS buffer solution was
used and partial specific volume was calculated based amino acid
compositions (V-bar of 0.712 mL/g and 0.722 mL/g for soluble HuJAM3
and soluble HuJAM2). The apparent molecular weights measured are
summarized in the Table 4 below and compared to that measured by
MALDI mass spectrometry. The difference between the calculated
molecular weight and that determined by MALDI indicates that the
protein is glycosylated. Soluble HuJAM2 contains two potential
N-linked glycosylation sites and soluble HuJAM3 contains 3
potential N-linked sites. These sites can be fully or partially
glycosylated when the protein is expressed from a mammalian cell
line. The molecular weight determined by AUC indicates that both
soluble HuJAM2 and soluble HuJAM3 are dimers in solution.
4TABLE 4 Molecular weight Calculated MW MW by MALDI MW by AUC
Sample (Da) (Da) (Da) Human LP121 23995 28295 60000 Human LP10034
26135 30177 58637
[0280] Size-exclusion chromatography was performed using a TSK-3000
column coupled with an on-line light scattering detector, miniDAWN
(Wyatt Technology Corp). Phosphate buffer saline containing 0.5M
NaCl buffer at pH 7.4 with 0.0005% sodium azide was used as mobile
phase. The flow rate of 0.5 mL/min and run time of 35 min were
used. Forty microliters of protein sample (0.2 to 0.6 mg/mL) was
injected onto the column. Molecular weight of the peak was
determined using the software supplied for the instrument (ASTRA
4.73.04). Soluble HuJAM3 runs as dimer on the size-exclusion
column, while soluble HuJAM2 runs as a monomer under these
conditions suggesting the dimer form is not very stable and can be
dissociated into monomers under the conditions used for SE-HPLC
experiment. When two proteins were mixed in 1:1 molar ratio, the
monomeric peak for soluble HuJAM2 disappeared, indicating that
soluble HuJAM3 and soluble HuJAM2 can form heterodimer.
[0281] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims. It is to be
understood that no limitation with respect to the specific examples
presented is intended or should be inferred. The disclosure is
intended to cover by the appended claims modifications as fall
within the scope of the claims.
Sequence CWU 1
1
28 1 299 PRT Homo sapiens 1 Met Gly Thr Lys Ala Gln Val Glu Arg Lys
Leu Leu Cys Leu Phe Ile 1 5 10 15 Leu Ala Ile Leu Leu Cys Ser Leu
Ala Leu Gly Ser Val Thr Val His 20 25 30 Ser Ser Glu Pro Glu Val
Arg Ile Pro Glu Asn Asn Pro Val Lys Leu 35 40 45 Ser Cys Ala Tyr
Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe 50 55 60 Asp Gln
Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr 65 70 75 80
Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe 85
90 95 Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val
Ser 100 105 110 Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys
Leu Ile Val 115 120 125 Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile
Pro Ser Ser Ala Thr 130 135 140 Ile Gly Asn Arg Ala Val Leu Thr Cys
Ser Glu Gln Asp Gly Ser Pro 145 150 155 160 Pro Ser Glu Tyr Thr Trp
Phe Lys Asp Gly Ile Val Met Pro Thr Asn 165 170 175 Pro Lys Ser Thr
Arg Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro 180 185 190 Thr Thr
Gly Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly 195 200 205
Glu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser 210
215 220 Asn Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile
Val 225 230 235 240 Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile
Leu Val Phe Gly 245 250 255 Ile Trp Phe Ala Tyr Ser Arg Gly His Phe
Asp Arg Thr Lys Lys Gly 260 265 270 Thr Ser Ser Lys Lys Val Ile Tyr
Ser Gln Pro Ser Ala Arg Ser Glu 275 280 285 Gly Glu Phe Lys Gln Thr
Ser Ser Phe Leu Val 290 295 2 298 PRT Homo sapiens 2 Met Ala Arg
Arg Ser Arg His Arg Phe Leu Leu Leu Leu Leu Arg Tyr 1 5 10 15 Leu
Val Val Ala Leu Gly Tyr His Lys Ala Tyr Gly Phe Ser Ala Pro 20 25
30 Lys Asp Gln Gln Val Val Thr Ala Val Glu Tyr Gln Glu Ala Ile Leu
35 40 45 Ala Cys Lys Thr Pro Lys Lys Thr Val Ser Ser Arg Leu Glu
Trp Lys 50 55 60 Lys Leu Gly Arg Ser Val Ser Phe Val Tyr Tyr Gln
Gln Thr Leu Gln 65 70 75 80 Gly Asp Phe Lys Asn Arg Ala Glu Met Ile
Asp Phe Asn Ile Arg Ile 85 90 95 Lys Asn Val Thr Arg Ser Asp Ala
Gly Lys Tyr Arg Cys Glu Val Ser 100 105 110 Ala Pro Ser Glu Gln Gly
Gln Asn Leu Glu Glu Asp Thr Val Thr Leu 115 120 125 Glu Val Leu Val
Ala Pro Ala Val Pro Ser Cys Glu Val Pro Ser Ser 130 135 140 Ala Leu
Ser Gly Thr Val Val Glu Leu Arg Cys Gln Asp Lys Glu Gly 145 150 155
160 Asn Pro Ala Pro Glu Tyr Thr Trp Phe Lys Asp Gly Ile Arg Leu Leu
165 170 175 Glu Asn Pro Arg Leu Gly Ser Gln Ser Thr Asn Ser Ser Tyr
Thr Met 180 185 190 Asn Thr Lys Thr Gly Thr Leu Gln Phe Asn Thr Val
Ser Lys Leu Asp 195 200 205 Thr Gly Glu Tyr Ser Cys Glu Ala Arg Asn
Ser Val Gly Tyr Arg Arg 210 215 220 Cys Pro Gly Lys Arg Met Gln Val
Asp Asp Leu Asn Ile Ser Gly Ile 225 230 235 240 Ile Ala Ala Val Val
Val Val Ala Leu Val Ile Ser Val Cys Gly Leu 245 250 255 Gly Val Cys
Tyr Ala Gln Arg Lys Gly Tyr Phe Ser Lys Glu Thr Ser 260 265 270 Phe
Gln Lys Ser Asn Ser Ser Ser Lys Ala Thr Thr Met Ser Glu Asn 275 280
285 Asp Phe Lys His Thr Lys Ser Phe Ile Ile 290 295 3 310 PRT Homo
sapiens 3 Met Ala Leu Arg Arg Pro Pro Arg Leu Arg Leu Cys Ala Arg
Leu Pro 1 5 10 15 Asp Phe Phe Leu Leu Leu Leu Phe Arg Gly Cys Leu
Ile Gly Ala Val 20 25 30 Asn Leu Lys Ser Ser Asn Arg Thr Pro Val
Val Gln Glu Arg Glu Ser 35 40 45 Val Glu Leu Ser Cys Ile Ile Thr
Asp Ser Gln Thr Ser Asp Pro Arg 50 55 60 Ile Glu Trp Lys Lys Ile
Gln Asp Glu Gln Thr Thr Tyr Val Phe Phe 65 70 75 80 Asp Asn Lys Ile
Gln Gly Asp Leu Ala Gly Arg Ala Glu Ile Leu Gly 85 90 95 Lys Thr
Ser Leu Lys Ile Trp Asn Val Thr Arg Arg Asp Ser Ala Leu 100 105 110
Tyr Arg Cys Glu Val Val Ala Arg Asn Asp Arg Lys Glu Ile Asp Glu 115
120 125 Ile Val Ile Glu Leu Thr Val Trp Val Lys Pro Val Thr Pro Val
Cys 130 135 140 Arg Val Pro Lys Ala Val Pro Val Gly Lys Met Ala Thr
Leu His Cys 145 150 155 160 Gln Glu Ser Glu Gly His Pro Arg Pro His
Tyr Ser Trp Tyr Arg Asn 165 170 175 Asp Val Pro Leu Pro Thr Asp Ser
Arg Ala Asn Pro Arg Phe Arg Asn 180 185 190 Ser Ser Phe His Leu Asn
Ser Glu Thr Gly Thr Leu Val Phe Thr Ala 195 200 205 Val His Lys Asp
Asp Ser Gly Gln Tyr Tyr Cys Ile Ala Ser Asn Asp 210 215 220 Ala Gly
Ser Ala Arg Cys Glu Glu Gln Glu Met Glu Val Tyr Asp Leu 225 230 235
240 Asn Ile Gly Gly Ile Ile Gly Gly Val Leu Val Val Leu Ala Val Leu
245 250 255 Ala Leu Ile Thr Leu Gly Ile Cys Cys Ala Tyr Arg Arg Gly
Tyr Phe 260 265 270 Ile Asn Asn Lys Gln Asp Gly Glu Ser Tyr Lys Asn
Pro Gly Lys Pro 275 280 285 Asp Gly Val Asn Tyr Ile Arg Thr Asp Glu
Glu Gly Asp Phe Arg His 290 295 300 Lys Ser Ser Phe Val Ile 305 310
4 900 DNA Homo sapiens 4 atggggacaa aggcgcaagt cgagaggaaa
ctgttgtgcc tcttcatatt ggcgatcctg 60 ttgtgctccc tggcattggg
cagtgttaca gtgcactctt ctgaacctga agtcagaatt 120 cctgagaata
atcctgtgaa gttgtcctgt gcctactcgg gcttttcttc tccccgtgtg 180
gagtggaagt ttgaccaagg agacaccacc agactcgttt gctataataa caagatcaca
240 gcttcctatg aggaccgggt gaccttcttg ccaactggta tcaccttcaa
gtccgtgaca 300 cgggaagaca ctgggacata cacttgtatg gtctctgagg
aaggcggcaa cagctatggg 360 gaggtcaagg tcaagctcat cgtgcttgtg
cctccatcca agcctacagt taacatcccc 420 tcctctgcca ccattgggaa
ccgggcagtg ctgacatgct cagaacaaga tggttcccca 480 ccttctgaat
acacctggtt caaagatggg atagtgatgc ctacgaatcc caaaagcacc 540
cgtgccttca gcaactcttc ctatgtcctg aatcccacaa caggagagct ggtctttgat
600 cccctgtcag cctctgatac tggagaatac agctgtgagg cacggaatgg
gtatgggaca 660 cccatgactt caaatgctgt gcgcatggaa gctgtggagc
ggaatgtggg ggtcatcgtg 720 gcagccgtcc ttgtaaccct gattctcctg
ggaatcttgg tttttggcat ctggtttgcc 780 tatagccgag gccactttga
cagaacaaag aaagggactt cgagtaagaa ggtgatttac 840 agccagccta
gtgcccgaag tgaaggagaa ttcaaacaga cctcgtcatt cctggtgtga 900 5 897
DNA Homo sapiens 5 atggcgagga ggagccgcca ccgcttcctc ctgctgctgc
tgcgctacct ggtggtcgcc 60 ctgggctatc ataaggccta tgggttttct
gccccaaaag accaacaggt agtcacagca 120 gtagagtacc aagaggctat
tttagcctgc aaaaccccaa agaagactgt ttcctccaga 180 ttagagtgga
agaaactggg tcggagtgtc tcctttgtct actatcaaca gactcttcaa 240
ggtgatttta aaaatcgagc tgagatgata gatttcaata tccggatcaa aaatgtgaca
300 agaagtgatg cggggaaata tcgttgtgaa gttagtgccc catctgagca
aggccaaaac 360 ctggaagagg atacagtcac tctggaagta ttagtggctc
cagcagttcc atcatgtgaa 420 gtaccctctt ctgctctgag tggaactgtg
gtagagctac gatgtcaaga caaagaaggg 480 aatccagctc ctgaatacac
atggtttaag gatggcatcc gtttgctaga aaatcccaga 540 cttggctccc
aaagcaccaa cagctcatac acaatgaata caaaaactgg aactctgcaa 600
tttaatactg tttccaaact ggacactgga gaatattcct gtgaagcccg caattctgtt
660 ggatatcgca ggtgtcctgg gaaacgaatg caagtagatg atctcaacat
aagtggcatc 720 atagcagccg tagtagttgt ggccttagtg atttccgttt
gtggccttgg tgtatgctat 780 gctcagagga aaggctactt ttcaaaagaa
acctccttcc agaagagtaa ttcttcatct 840 aaagccacga caatgagtga
aaatgatttc aagcacacaa aatcctttat aatttaa 897 6 933 DNA Homo sapiens
6 atggcgctga ggcggccacc gcgactccgg ctctgcgctc ggctgcctga cttcttcctg
60 ctgctgcttt tcaggggctg cctgataggg gctgtaaatc tcaaatccag
caatcgaacc 120 ccagtggtac aggaatttga aagtgtggaa ctgtcttgca
tcattacgga ttcgcagaca 180 agtgacccca ggatcgagtg gaagaaaatt
caagatgaac aaaccacata tgtgtttttt 240 gacaacaaaa ttcagggaga
cttggcgggt cgtgcagaaa tactggggaa gacatccctg 300 aagatctgga
atgtgacacg gagagactca gccctttatc gctgtgaggt cgttgctcga 360
aatgaccgca aggaaattga tgagattgtg atcgagttaa ctgtgcaagt gaagccagtg
420 acccctgtct gtagagtgcc gaaggctgta ccagtaggca agatggcaac
actgcactgc 480 caggagagtg agggccaccc ccggcctcac tacagctggt
atcgcaatga tgtaccactg 540 cccacggatt ccagagccaa tcccagattt
cgcaattctt cttcccactt aaactctgaa 600 acaggcactt tggtgttcac
tgctgttcac aaggacgact ctgggcagta ctactgcatt 660 gcttccaatg
acgcaggctc agccaggtgt gaggagcagg agatggaagt ctatgacctg 720
aacattggcg gaattattgg gggggttctg gttgtccttg ctgtactggc cctgatcacg
780 ttgggcatct gctgtgcata cagacgtggc tacttcatca acaataaaca
ggatggagaa 840 agttacaaga acccagggaa accagatgga gttaactaca
tccgcactga cgaggagggc 900 gacttcagac acaagtcatc gtttgtgatc tga 933
7 235 PRT Homo sapiens 7 Met Gly Thr Lys Ala Gln Val Glu Arg Lys
Leu Leu Cys Leu Phe Ile 1 5 10 15 Leu Ala Ile Leu Leu Cys Ser Leu
Ala Leu Gly Ser Val Thr Val His 20 25 30 Ser Ser Glu Pro Glu Val
Arg Ile Pro Glu Asn Asn Pro Val Lys Leu 35 40 45 Ser Cys Ala Tyr
Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe 50 55 60 Asp Gln
Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr 65 70 75 80
Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe 85
90 95 Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val
Ser 100 105 110 Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys
Leu Ile Val 115 120 125 Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile
Pro Ser Ser Ala Thr 130 135 140 Ile Gly Asn Arg Ala Val Leu Thr Cys
Ser Glu Gln Asp Gly Ser Pro 145 150 155 160 Pro Ser Glu Tyr Thr Trp
Phe Lys Asp Gly Ile Val Met Pro Thr Asn 165 170 175 Pro Lys Ser Thr
Arg Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro 180 185 190 Thr Thr
Gly Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly 195 200 205
Glu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser 210
215 220 Asn Ala Val Arg Met Glu Ala Val Glu Arg Asn 225 230 235 8
208 PRT Homo sapiens 8 Ser Val Thr Val His Ser Ser Glu Pro Glu Val
Arg Ile Pro Glu Asn 1 5 10 15 Asn Pro Val Lys Leu Ser Cys Ala Tyr
Ser Gly Phe Ser Ser Pro Arg 20 25 30 Val Glu Trp Lys Phe Asp Gln
Gly Asp Thr Thr Arg Leu Val Cys Tyr 35 40 45 Asn Asn Lys Ile Thr
Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro 50 55 60 Thr Gly Ile
Thr Phe Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr 65 70 75 80 Thr
Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys 85 90
95 Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile
100 105 110 Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val Leu Thr Cys
Ser Glu 115 120 125 Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp Phe
Lys Asp Gly Ile 130 135 140 Val Met Pro Thr Asn Pro Lys Ser Thr Arg
Ala Phe Ser Asn Ser Ser 145 150 155 160 Tyr Val Leu Asn Pro Thr Thr
Gly Glu Leu Val Phe Asp Pro Leu Ser 165 170 175 Ala Ser Asp Thr Gly
Glu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly 180 185 190 Thr Pro Met
Thr Ser Asn Ala Val Arg Met Glu Ala Val Glu Arg Asn 195 200 205 9
223 PRT Homo sapiens 9 Met Ala Arg Arg Ser Arg His Arg Phe Leu Leu
Leu Leu Leu Arg Tyr 1 5 10 15 Leu Val Val Ala Leu Gly Tyr His Lys
Ala Tyr Gly Phe Ser Ala Pro 20 25 30 Lys Asp Gln Gln Val Val Thr
Ala Val Glu Tyr Gln Glu Ala Ile Leu 35 40 45 Ala Cys Lys Thr Pro
Lys Lys Thr Val Ser Ser Arg Leu Glu Trp Lys 50 55 60 Lys Leu Gly
Arg Ser Val Ser Phe Val Tyr Tyr Gln Gln Thr Leu Gln 65 70 75 80 Gly
Asp Phe Lys Asn Arg Ala Glu Met Ile Asp Phe Asn Ile Arg Ile 85 90
95 Lys Asn Val Thr Arg Ser Asp Ala Gly Lys Tyr Arg Cys Glu Val Ser
100 105 110 Ala Pro Ser Glu Gln Gly Gln Asn Leu Glu Glu Asp Thr Val
Thr Leu 115 120 125 Glu Val Leu Val Ala Pro Ala Val Pro Ser Cys Glu
Val Pro Ser Ser 130 135 140 Ala Leu Ser Gly Thr Val Val Glu Leu Arg
Cys Gln Asp Lys Glu Gly 145 150 155 160 Asn Pro Ala Pro Glu Tyr Thr
Trp Phe Lys Asp Gly Ile Arg Leu Leu 165 170 175 Glu Asn Pro Arg Leu
Gly Ser Gln Ser Thr Asn Ser Ser Tyr Thr Met 180 185 190 Asn Thr Lys
Thr Gly Thr Leu Gln Phe Asn Thr Val Ser Lys Leu Asp 195 200 205 Thr
Gly Glu Tyr Ser Cys Glu Ala Arg Asn Ser Val Gly Tyr Arg 210 215 220
10 236 PRT Homo sapiens 10 Met Ala Arg Arg Ser Arg His Arg Phe Leu
Leu Leu Leu Leu Arg Tyr 1 5 10 15 Leu Val Val Ala Leu Gly Tyr His
Lys Ala Tyr Gly Phe Ser Ala Pro 20 25 30 Lys Asp Gln Gln Val Val
Thr Ala Val Glu Tyr Gln Glu Ala Ile Leu 35 40 45 Ala Cys Lys Thr
Pro Lys Lys Thr Val Ser Ser Arg Leu Glu Trp Lys 50 55 60 Lys Leu
Gly Arg Ser Val Ser Phe Val Tyr Tyr Gln Gln Thr Leu Gln 65 70 75 80
Gly Asp Phe Lys Asn Arg Ala Glu Met Ile Asp Phe Asn Ile Arg Ile 85
90 95 Lys Asn Val Thr Arg Ser Asp Ala Gly Lys Tyr Arg Cys Glu Val
Ser 100 105 110 Ala Pro Ser Glu Gln Gly Gln Asn Leu Glu Glu Asp Thr
Val Thr Leu 115 120 125 Glu Val Leu Val Ala Pro Ala Val Pro Ser Cys
Glu Val Pro Ser Ser 130 135 140 Ala Leu Ser Gly Thr Val Val Glu Leu
Arg Cys Gln Asp Lys Glu Gly 145 150 155 160 Asn Pro Ala Pro Glu Tyr
Thr Trp Phe Lys Asp Gly Ile Arg Leu Leu 165 170 175 Glu Asn Pro Arg
Leu Gly Ser Gln Ser Thr Asn Ser Ser Tyr Thr Met 180 185 190 Asn Thr
Lys Thr Gly Thr Leu Gln Phe Asn Thr Val Ser Lys Leu Asp 195 200 205
Thr Gly Glu Tyr Ser Cys Glu Ala Arg Asn Ser Val Gly Tyr Arg Arg 210
215 220 Cys Pro Gly Lys Arg Met Gln Val Asp Asp Leu Asn 225 230 235
11 196 PRT Homo sapiens 11 Phe Ser Ala Pro Lys Asp Gln Gln Val Val
Thr Ala Val Glu Tyr Gln 1 5 10 15 Glu Ala Ile Leu Ala Cys Lys Thr
Pro Lys Lys Thr Val Ser Ser Arg 20 25 30 Leu Glu Trp Lys Lys Leu
Gly Arg Ser Val Ser Phe Val Tyr Tyr Gln 35 40 45 Gln Thr Leu Gln
Gly Asp Phe Lys Asn Arg Ala Glu Met Ile Asp Phe 50 55 60 Asn Ile
Arg Ile Lys Asn Val Thr Arg Ser Asp Ala Gly Lys Tyr Arg 65 70 75 80
Cys Glu Val Ser Ala Pro Ser Glu Gln Gly Gln Asn Leu Glu Glu Asp 85
90 95 Thr Val Thr Leu Glu Val Leu Val Ala Pro Ala Val Pro Ser Cys
Glu 100 105 110 Val Pro Ser Ser Ala Leu Ser Gly Thr Val Val Glu Leu
Arg Cys Gln 115 120 125 Asp Lys Glu Gly Asn Pro Ala Pro Glu Tyr Thr
Trp Phe Lys Asp Gly 130 135 140 Ile Arg Leu Leu Glu Asn Pro Arg Leu
Gly Ser Gln Ser Thr Asn Ser 145 150
155 160 Ser Tyr Thr Met Asn Thr Lys Thr Gly Thr Leu Gln Phe Asn Thr
Val 165 170 175 Ser Lys Leu Asp Thr Gly Glu Tyr Ser Cys Glu Ala Arg
Asn Ser Val 180 185 190 Gly Tyr Arg Arg 195 12 208 PRT Homo sapiens
12 Phe Ser Ala Pro Lys Asp Gln Gln Val Val Thr Ala Val Glu Tyr Gln
1 5 10 15 Glu Ala Ile Leu Ala Cys Lys Thr Pro Lys Lys Thr Val Ser
Ser Arg 20 25 30 Leu Glu Trp Lys Lys Leu Gly Arg Ser Val Ser Phe
Val Tyr Tyr Gln 35 40 45 Gln Thr Leu Gln Gly Asp Phe Lys Asn Arg
Ala Glu Met Ile Asp Phe 50 55 60 Asn Ile Arg Ile Lys Asn Val Thr
Arg Ser Asp Ala Gly Lys Tyr Arg 65 70 75 80 Cys Glu Val Ser Ala Pro
Ser Glu Gln Gly Gln Asn Leu Glu Glu Asp 85 90 95 Thr Val Thr Leu
Glu Val Leu Val Ala Pro Ala Val Pro Ser Cys Glu 100 105 110 Val Pro
Ser Ser Ala Leu Ser Gly Thr Val Val Glu Leu Arg Cys Gln 115 120 125
Asp Lys Glu Gly Asn Pro Ala Pro Glu Tyr Thr Trp Phe Lys Asp Gly 130
135 140 Ile Arg Leu Leu Glu Asn Pro Arg Leu Gly Ser Gln Ser Thr Asn
Ser 145 150 155 160 Ser Tyr Thr Met Asn Thr Lys Thr Gly Thr Leu Gln
Phe Asn Thr Val 165 170 175 Ser Lys Leu Asp Thr Gly Glu Tyr Ser Cys
Glu Ala Arg Asn Ser Val 180 185 190 Gly Tyr Arg Arg Cys Pro Gly Lys
Arg Met Gln Val Asp Asp Leu Asn 195 200 205 13 238 PRT Homo sapiens
13 Met Ala Leu Arg Arg Pro Pro Arg Leu Arg Leu Cys Ala Arg Leu Pro
1 5 10 15 Asp Phe Phe Leu Leu Leu Leu Phe Arg Gly Cys Leu Ile Gly
Ala Val 20 25 30 Asn Leu Lys Ser Ser Asn Arg Thr Pro Val Val Gln
Glu Phe Glu Ser 35 40 45 Val Glu Leu Ser Cys Ile Ile Thr Asp Ser
Gln Thr Ser Asp Pro Arg 50 55 60 Ile Glu Trp Lys Lys Ile Gln Asp
Glu Gln Thr Thr Tyr Val Phe Phe 65 70 75 80 Asp Asn Lys Ile Gln Gly
Asp Leu Ala Gly Arg Ala Glu Ile Leu Gly 85 90 95 Lys Thr Ser Leu
Lys Ile Trp Asn Thr Arg Arg Asp Ser Ala Leu Arg 100 105 110 Cys Glu
Val Val Ala Arg Asn Asp Arg Lys Glu Ile Asp Glu Ile Val 115 120 125
Ile Glu Leu Thr Val Gln Val Lys Pro Val Thr Pro Val Cys Arg Val 130
135 140 Pro Lys Ala Val Pro Val Gly Lys Met Ala Thr Leu His Cys Gln
Glu 145 150 155 160 Ser Glu Gly His Pro Arg Pro His Tyr Ser Trp Tyr
Arg Asn Asp Val 165 170 175 Pro Leu Pro Thr Asp Ser Arg Ala Asn Pro
Arg Phe Arg Asn Ser Ser 180 185 190 Ser His Leu Asn Ser Glu Thr Gly
Thr Leu Val Phe Thr Ala Val His 195 200 205 Lys Asp Asp Ser Gly Gln
Tyr Tyr Cys Ile Ala Ser Asn Asp Ala Gly 210 215 220 Ser Ala Arg Cys
Glu Glu Gln Glu Met Glu Val Tyr Asp Leu 225 230 235 14 210 PRT Homo
sapiens 14 Ala Val Asn Leu Lys Ser Ser Asn Arg Thr Pro Val Val Gln
Glu Phe 1 5 10 15 Glu Ser Val Glu Leu Ser Cys Ile Ile Thr Asp Ser
Gln Thr Ser Asp 20 25 30 Pro Arg Ile Glu Trp Lys Lys Ile Gln Asp
Glu Gln Thr Thr Tyr Val 35 40 45 Phe Phe Asp Asn Lys Ile Gln Gly
Asp Leu Ala Gly Arg Ala Glu Ile 50 55 60 Leu Gly Lys Thr Ser Leu
Lys Ile Trp Asn Val Thr Arg Arg Asp Ser 65 70 75 80 Ala Leu Tyr Arg
Cys Glu Val Val Ala Arg Asn Asp Arg Lys Glu Ile 85 90 95 Asp Glu
Ile Val Ile Glu Leu Thr Val Gln Val Lys Pro Val Thr Pro 100 105 110
Val Cys Arg Val Pro Lys Ala Val Pro Val Gly Lys Met Ala Thr Leu 115
120 125 His Cys Gln Glu Ser Glu Gly His Pro Arg Pro His Tyr Ser Trp
Tyr 130 135 140 Arg Asn Asp Val Pro Leu Pro Thr Asp Ser Arg Ala Asn
Pro Arg Phe 145 150 155 160 Arg Asn Ser Ser Ser His Leu Asn Ser Glu
Thr Gly Thr Leu Val Phe 165 170 175 Thr Ala Val His Lys Asp Asp Ser
Gly Gln Tyr Tyr Cys Ile Ala Ser 180 185 190 Asn Asp Ala Gly Ser Ala
Arg Cys Glu Glu Gln Glu Met Glu Val Tyr 195 200 205 Asp Leu 210 15
705 DNA Homo sapiens 15 atggggacaa aggcgcaagt cgagaggaaa ctgttgtgcc
tcttcatatt ggcgatcctg 60 ttgtgctccc tggcattggg cagtgttaca
gtgcactctt ctgaacctga agtcagaatt 120 cctgagaata atcctgtgaa
gttgtcctgt gcctactcgg gcttttcttc tccccgtgtg 180 gagtggaagt
ttgaccaagg agacaccacc agactcgttt gctataataa caagatcaca 240
gcttcctatg aggaccgggt gaccttcttg ccaactggta tcaccttcaa gtccgtgaca
300 cgggaagaca ctgggacata cacttgtatg gtctctgagg aaggcggcaa
cagctatggg 360 gaggtcaagg tcaagctcat cgtgcttgtg cctccatcca
agcctacagt taacatcccc 420 tcctctgcca ccattgggaa ccgggcagtg
ctgacatgct cagaacaaga tggttcccca 480 ccttctgaat acacctggtt
caaagatggg atagtgatgc ctacgaatcc caaaagcacc 540 cgtgccttca
gcaactcttc ctatgtcctg aatcccacaa caggagagct ggtctttgat 600
cccctgtcag cctctgatac tggagaatac agctgtgagg cacggaatgg gtatgggaca
660 cccatgactt caaatgctgt gcgcatggaa gctgtggagc ggaat 705 16 672
DNA Homo sapiens 16 atggcgagga ggagccgcca ccgcttcctc ctgctgctgc
tgcgctacct ggtggtcgcc 60 ctgggctatc ataaggccta tgggttttct
gccccaaaag accaacaggt agtcacagca 120 gtagagtacc aagaggctat
tttagcctgc aaaaccccaa agaagactgt ttcctccaga 180 ttagagtgga
agaaactggg tcggagtgtc tcctttgtct actatcaaca gactcttcaa 240
ggtgatttta aaaatcgagc tgagatgata gatttcaata tccggatcaa aaatgtgaca
300 agaagtgatg cggggaaata tcgttgtgaa gttagtgccc catctgagca
aggccaaaac 360 ctggaagagg atacagtcac tctggaagta ttagtggctc
cagcagttcc atcatgtgaa 420 gtaccctctt ctgctctgag tggaactgtg
gtagagctac gatgtcaaga caaagaaggg 480 aatccagctc ctgaatacac
atggtttaag gatggcatcc gtttgctaga aaatcccaga 540 cttggctccc
aaagcaccaa cagctcatac acaatgaata caaaaactgg aactctgcaa 600
tttaatactg tttccaaact ggacactgga gaatattcct gtgaagcccg caattctgtt
660 ggatatcgca gg 672 17 708 DNA Homo sapiens 17 atggcgagga
ggagccgcca ccgcttcctc ctgctgctgc tgcgctacct ggtggtcgcc 60
ctgggctatc ataaggccta tgggttttct gccccaaaag accaacaggt agtcacagca
120 gtagagtacc aagaggctat tttagcctgc aaaaccccaa agaagactgt
ttcctccaga 180 ttagagtgga agaaactggg tcggagtgtc tcctttgtct
actatcaaca gactcttcaa 240 ggtgatttta aaaatcgagc tgagatgata
gatttcaata tccggatcaa aaatgtgaca 300 agaagtgatg cggggaaata
tcgttgtgaa gttagtgccc catctgagca aggccaaaac 360 ctggaagagg
atacagtcac tctggaagta ttagtggctc cagcagttcc atcatgtgaa 420
gtaccctctt ctgctctgag tggaactgtg gtagagctac gatgtcaaga caaagaaggg
480 aatccagctc ctgaatacac atggtttaag gatggcatcc gtttgctaga
aaatcccaga 540 cttggctccc aaagcaccaa cagctcatac acaatgaata
caaaaactgg aactctgcaa 600 tttaatactg tttccaaact ggacactgga
gaatattcct gtgaagcccg caattctgtt 660 ggatatcgca ggtgtcctgg
gaaacgaatg caagtagatg atctcaac 708 18 720 DNA Homo sapiens 18
atggcgctga ggcggccacc gcgactccgg ctctgcgctc ggctgcctga cttcttcctg
60 ctgctgcttt tcaggggctg cctgataggg gctgtaaatc tcaaatccag
caatcgaacc 120 ccagtggtac aggaatttga aagtgtggaa ctgtcttgca
tcattacgga ttcgcagaca 180 agtgacccca ggatcgagtg gaagaaaatt
caagatgaac aaaccacata tgtgtttttt 240 gacaacaaaa ttcagggaga
cttggcgggt cgtgcagaaa tactggggaa gacatccctg 300 aagatctgga
atgtgacacg gagagactca gccctttatc gctgtgaggt cgttgctcga 360
aatgaccgca aggaaattga tgagattgtg atcgagttaa ctgtgcaagt gaagccagtg
420 acccctgtct gtagagtgcc gaaggctgta ccagtaggca agatggcaac
actgcactgc 480 caggagagtg agggccaccc ccggcctcac tacagctggt
atcgcaatga tgtaccactg 540 cccacggatt ccagagccaa tcccagattt
cgcaattctt cttcccactt aaactctgaa 600 acaggcactt tggtgttcac
tgctgttcac aaggacgact ctgggcagta ctactgcatt 660 gcttccaatg
acgcaggctc agccaggtgt gaggagcagg agatggaagt ctatgacctg 720 19 624
DNA Homo sapiens 19 agtgttacag tgcactcttc tgaacctgaa gtcagaattc
ctgagaataa tcctgtgaag 60 ttgtcctgtg cctactcggg cttttcttct
ccccgtgtgg agtggaagtt tgaccaagga 120 gacaccacca gactcgtttg
ctataataac aagatcacag cttcctatga ggaccgggtg 180 accttcttgc
caactggtat caccttcaag tccgtgacac gggaagacac tgggacatac 240
acttgtatgg tctctgagga aggcggcaac agctatgggg aggtcaaggt caagctcatc
300 gtgcttgtgc ctccatccaa gcctacagtt aacatcccct cctctgccac
cattgggaac 360 cgggcagtgc tgacatgctc agaacaagat ggttccccac
cttctgaata cacctggttc 420 aaagatggga tagtgatgcc tacgaatccc
aaaagcaccc gtgccttcag caactcttcc 480 tatgtcctga atcccacaac
aggagagctg gtctttgatc ccctgtcagc ctctgatact 540 ggagaataca
gctgtgaggc acggaatggg tatgggacac ccatgacttc aaatgctgtg 600
cgcatggaag ctgtggagcg gaat 624 20 587 DNA Homo sapiens 20
ttttctgccc caaaagacca acaggtagtc acagcagtag agtaccaaga ggctatttta
60 gcctgcaaaa ccccaaagaa gactgtttcc tccagattag agtggaagaa
actgggtcgg 120 agtgtctcct ttgtctacta tcaacagact cttcaaggtg
attttaaaaa tcgagctgag 180 atgatagatt tcaatatccg gatcaaaaat
gtgacaagaa gtgatgcggg gaaatatcgt 240 tgtgaagtta gtgccccatc
tgagcaaggc caaaacctgg aagaggatac agtcactctg 300 gaagtattag
tggctccagc agttccatca tgtgaagtac cctcttctgc tctgagtgga 360
actgtggtag agctacgatg tcaagacaaa gaagggaatc cagctcctga atacacatgg
420 tttaaggatg gcatccgttt gctagaaaat cccagacttg gctcccaaag
caccaacagc 480 tcatacacaa tgaatacaaa aactggaact ctgcaattta
atactgtttc caaactggac 540 actgggaata ttcctgtgaa gcccgcaatt
ctgttggata tcgcagg 587 21 623 DNA Homo sapiens 21 ttttctgccc
caaaagacca acaggtagtc acagcagtag agtaccaaga ggctatttta 60
gcctgcaaaa ccccaaagaa gactgtttcc tccagattag agtggaagaa actgggtcgg
120 agtgtctcct ttgtctacta tcaacagact cttcaaggtg attttaaaaa
tcgagctgag 180 atgatagatt tcaatatccg gatcaaaaat gtgacaagaa
gtgatgcggg gaaatatcgt 240 tgtgaagtta gtgccccatc tgagcaaggc
caaaacctgg aagaggatac agtcactctg 300 gaagtattag tggctccagc
agttccatca tgtgaagtac cctcttctgc tctgagtgga 360 actgtggtag
agctacgatg tcaagacaaa gaagggaatc cagctcctga atacacatgg 420
tttaaggatg gcatccgttt gctagaaaat cccagacttg gctcccaaag caccaacagc
480 tcatacacaa tgaatacaaa aactggaact ctgcaattta atactgtttc
caaactggac 540 actgggaata ttcctgtgaa gcccgcaatt ctgttggata
tcgcaggtgt cctgggaaac 600 gaatgcaagt agatgatctc aac 623 22 630 DNA
Homo sapiens 22 gctgtaaatc tcaaatccag caatcgaacc ccagtggtac
aggaatttga aagtgtggaa 60 ctgtcttgca tcattacgga ttcgcagaca
agtgacccca ggatcgagtg gaagaaaatt 120 caagatgaac aaaccacata
tgtgtttttt gacaacaaaa ttcagggaga cttggcgggt 180 cgtgcagaaa
tactggggaa gacatccctg aagatctgga atgtgacacg gagagactca 240
gccctttatc gctgtgaggt cgttgctcga aatgaccgca aggaaattga tgagattgtg
300 atcgagttaa ctgtgcaagt gaagccagtg acccctgtct gtagagtgcc
gaaggctgta 360 ccagtaggca agatggcaac actgcactgc caggagagtg
agggccaccc ccggcctcac 420 tacagctggt atcgcaatga tgtaccactg
cccacggatt ccagagccaa tcccagattt 480 cgcaattctt cttcccactt
aaactctgaa acaggcactt tggtgttcac tgctgttcac 540 aaggacgact
ctgggcagta ctactgcatt gcttccaatg acgcaggctc agccaggtgt 600
gaggagcagg agatggaagt ctatgacctg 630 23 36 DNA Artificial Sequence
Synthetic Construct 23 ttactaggct ggcgcgccac catggcgagg aggagc 36
24 35 DNA Artificial Sequence Synthetic Construct 24 aggaaggaat
gcgcaaatta taaaggattt tgtgt 35 25 37 DNA Artificial Sequence
Synthetic Construct 25 gatcggcgcg ccagccacca tggcgctgag gcggcca 37
26 33 DNA Artificial Sequence Synthetic Construct 26 cgccggttta
aacgccaggt catagacttc cat 33 27 18 DNA Artificial Sequence
Synthetic Construct 27 tgcaaagctt ggcgcgcc 18 28 25 DNA Artificial
Sequence Synthetic Construct 28 cttgtcgtcg tcatccttgt agtcg 25
* * * * *